Pharmaceutical composition comprising oxyntomodulin derivatives and a method for reducing body weight using the composition

ABSTRACT

Modified oxyntomodulin derivatives. Such derivatives can be used for the treatment of metabolic diseases such as diabetes and obesity.

CROSS REFERENCE TO RELATED APPLICATIONS

This patent application claims benefit of International PatentApplication No. PCT/US2007/004306, which was filed 16 Feb. 2007 andwhich claims benefit of U.S. Provisional Application No. 60/775,544,filed 22 Feb. 2006, and U.S. Provisional Application No. 60/834,452,filed 31 Jul. 2006.

FIELD OF THE INVENTION

The present invention relates to oxyntomodulin derivatives, theirsynthesis, and their use for the treatment of metabolic disorders suchas diabetes and obesity.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The sequence listing of the present application is submittedelectronically via EFS-Web as an ASCII formatted sequence listing with afile name “MRLDOB22046USPCT-SEQTXT-30APR2010.txt”, creation date of Apr.30, 2010, and a size of 114 KB. This sequence listing submitted viaEFS-Web is part of the specification and is herein incorporated byreference in its entirety.

BACKGROUND OF THE INVENTION

The hormone oxyntomodulin (OXM, glucagon-37) is a posttranslationalproduct of preproglucagon processing in the intestine and centralnervous system (CNS) and is secreted from L-cells in the gut in responseto food intake. Discovered in 1983, OXM has been implicated in theregulation of food intake and energy expenditure. Central or peripheraladministration of OXM in rats causes a decrease in short term foodintake with minimal effects on gastric emptying (Dakin et al.Endocrinology, 142:4244-4250 (2001), Dakin et al. Endocrinology,145:2687-2695 (2004)). Repeated intracerebroventricular administrationof OXM in rats results in elevated core temperatures and reduced weightgain compared to pair-fed animals, suggesting effects on both caloricintake and energy expenditure (Dakin et al. Am. J. Physiol. Endocrinol.Metab., 283:E1173-E1177 (2002)).

OXM is a 37-amino acid peptide. It has been reported that the effects ofOXM in inhibiting gastric acid secretion can be mimicked by the8-residue C-terminal fragment Oxm(30-37), known as SP-1 (Caries-Bonnetet al., Peptides, 1996, 17:557-561. In humans, a single 90 minintravenous infusion of OXM in normal weight healthy subjects reducedhunger scores and food intake at a buffet meal by ˜19%. Cumulative 12hour caloric intake was reduced by ˜11% with no reports of nausea orchanges in food palatability (Cohen et al., J. Clin. Endocrinol. Metab.,88:4696-4701 (2003)). More recently, pre-prandial injections of OXM overa 4 week period in obese healthy volunteers (BMI ˜33) led to asignificant reduction of caloric intake on the first day of treatment(˜25%) that was maintained over the course of the study (35% reductionafter 4 weeks) (Wynne et al., Diabetes 54:2390-2395 (2005)). Robustweight loss was observed at the end of the study in treated subjects(1.9%, placebo-corrected). Plasma levels of OXM were similar to thatobserved in the infusion study (peak concentration ˜950 pM). The absenceof any tachyphylaxis and a low incidence of mild and transient nausea(˜3%) despite the relatively high doses necessitated by the poor in vivostability of OXM (plasma t_(1/2)<12 min) renders this hormone one of thefew obesity targets with both human validation and an attractivetolerability profile.

OXM has a very short half-life and is rapidly inactivated by the cellsurface dipeptidyl peptidase IV (hereafter DP-IV). However, DP-IVinhibitors are weight-neutral in the clinic, suggesting thatsupraphysiological levels of OXM (900-1000 pM) may be required toachieve weight loss in humans.

Oxyntomodulin therefore shows potential as a treatment for metabolicdisorders such as diabetes and obesity. However, because of the poor invivo stability of OXM, there exists a need to develop OXM derivativesthat can be safely and efficaciously administered for the treatment ofmetabolic diseases, such as diabetes and obesity. It would be furtherdesirable if analogs or derivatives were developed that were modified byconjugation to moieties that would improve stability andpharmacokinetics, more particularly modifications that confer resistanceto DP-IV cleavage. The instant invention provides OXM polypeptidederivatives and methods for the treatment or prevention of metabolicdisorders such as obesity and diabetes by administering the derivativesdescribed herein.

SUMMARY OF THE INVENTION

The present invention provides a polypeptide comprising:

(SEQ ID NO: 160) H_(x)X₁X₂GTFTSDYX₃X₄YLDX₅X₆X₆AX₇X₈FVX₇WLX₉X₁₀X₁₁KRNRNNX₁₂X₁₃X₁₄,

wherein H_(x) is selected from the group consisting of His;imidazole-lactic acid (ImiH); desamino-His (ΔNH₂-H); acetyl His;pyroglutamyl His (PyrH); N-methyl-His (Me-H); N,N-dimethyl-His (Me₂-H);Benzoyl His (Bz-H); Benzyl His (Bzl-H); and Phe;

X₁ is selected from the group consisting of Ser; Gly; Ala; Arg; Asn;Asp; Glu; Gln; His; Ile; Lys; Met; Phe; Pro; Thr; Trp; Tyr; Val; D-Ala;D-Ser; and α-aminoisobutyric acid;

X₂ is Gln, Asp, Glu, Pro, Leu or L-norleucine;

X₃ is Ser, Ala, Cys, Cys(mPEG), or Cys(cholesteryl);

X₄ is Lys, Cys, Cys(mPEG), or Cys(cholesteryl);

X₅ is Ser or Ala;

X₆ is any amino acid;

X₇ is Gln, Cys, Cys(mPEG), or Cys(cholesteryl);

X₈ is Asp, Cys, Cys(mPEG), or Cys(cholesteryl);

X₉ is Met, Met(O), Val, norleucine, alanine, α-aminoisobutyric acid orO-methyl-homoserine;

X₁₀ is Asn, Cys, Cys(mPEG), or Cys(cholesteryl);

X₁₁ is Thr, Cys, Cys(mPEG), or Cys(cholesteryl);

X₁₂ is Ile, Cys, Cys(mPEG), or Cys(cholesteryl);

X₁₃ is Ala, Cys, Cys(mPEG), or Cys(cholesteryl); and

X₁₄ is amide, carboxylate, secondary amide, Ala, K(palmitoyl), Cys,Cys(mPEG), Cys(cholesteryl) or any linker to which mPEG or cholesterolis linked with a chemical bond. Pharmaceutically acceptable saltsthereof are contemplated as well.

Additionally, any one or two of X₃, X₄, X₆-X₈, and X₁₀-X₁₄ may beCys(mPEG) Cys(cholesteryl); Cys(mPEG)teine may also be C₁; C₂; C₃ or C₆,wherein C₁=Cys(mPEG)₅ kDa, C₂=Cys(mPEG)20 kDa, C₃=Cys(mPEG)₂40 kDa,C₆=Cys(MPEG)₂60 kDa and each corresponds to a cysteine residue PEGylatedvia the side chain thiol with linear methoxyPEG (mPEG) or branched mPEG₂of the indicated molecular weight.

The present invention relates to OXM polypeptide derivatives of theformula:

(SEQ ID NO: 120) HαDGTFTSDYSKYLDSRRAQDFVQWLmNTKRNRNNIAC₃wherein C₃=Cys[(mPEG)₂40 kDa], each corresponding to an amidatedcysteine residue PEGylated via the side-chain thiol with a branched mPEG[(mPEG)₂] of the indicated MW; α is α-amino isobutyric acid (aib); andm=methionine sulfoxide (Met(O)).

In another embodiment of the present invention, there is provided apolypeptide comprising:

(SEQ ID NO: 161) H_(x)SQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA,wherein H_(x) is selected from the group consisting of His,H₁=Imidazole-lactic acid (ImiH); desamino-His (ΔNH₂-H), acetyl His,pyroglutamyl His, N-methyl-His (Me-H), N,N-dimethyl-His (Me₂-H); BenzoylHis (Bz-H), Benzyl His (Bzl-H), and Phe.

In yet another embodiment of the present invention, there is provided apolypeptide comprising:

(SEQ ID NO: 162) HX₁QGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA

wherein X₁ is selected from the group consisting of Ser, Gly, Ala, Arg;Asn, Asp, Glu, Gln, His, Ile, Lys, Met, Phe, Pro, Thr, Trp, Tyr, Val,D-Ala, D-Ser, and α-aminoisobutyric acid.

The present invention further provides for a polypeptide comprising:

(SEQ ID NO: 163) HSX₂GTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA

X₂ is selected from the group consisting of Gln, Asp, Gln, Pro, Len, andL-norleucine.

In another embodiment of the present invention, there is provided apolypeptide comprising:

(SEQ ID NO: 164) HSQGTFTSDYX₃X₄YLDSX₆X₆AX₇X₈FVX₇WLMX₁₀X₁₁KRNRNNX₁₂X₁₃X₁₄

wherein X₃ is Ser, Ala, Cys(mPEG), or Cys(cholesteryl);

X₄ is Lys, Cys(mPEG), or Cys(cholesteryl);

X₆ is any one of Arg, Cys(mPEG), or Cys(cholesteryl);

X₇ is any one of Gln, Cys(mPEG), or Cys(cholesteryl);

X₈ is Asp, Cys(mPEG), or Cys(cholesteryl);

X₁₀ is Asn, Cys(mPEG), or Cys(cholesteryl);

X₁₁ is Thr, Cys(mPEG), or Cys(cholesteryl);

X₁₂ is Ile, Cys(mPEG), or Cys(cholesteryl);

X₁₃ is Ala, Cys(mPEG), or Cys(cholesteryl); and

X₁₄ is amide, carboxylate, secondary amide, Ala, K(palmitoyl),Cys(mPEG), Cys(cholesteryl), or any linker to which mPEG or cholesterolis linked with a chemical bond.

wherein one or two of X₃ X₄, X₆-X₈, and X₁₀-X₁₄ is Cys(mPEG) orCys(cholesteryl).

In an embodiment of the present invention, there is provided apolypeptide comprising:

(SEQ ID NO: 165) HαX₁₅GTFTSDYSKYLDSZZAX₁₆DFVQWLX₁₇NTX₁₈

wherein X₁₅ is D or Q;

Z is any amino acid;

X₁₆ is C₈, Cys(N-ethylmaleimidyl), Q or C;

X₁₇ is m or M;

X₁₈ is an amidated k or K.

In another embodiment of the present invention, there is provided apolypeptide comprising:

(SEQ ID NO: 166) HαDGTFTSDYSKYDSZZAQDFVQWLmNTKRNRNNIAX₁₉,

wherein X₁₉ is C or C₈, Cys(N-ethylmaleimidyl).

In yet another embodiment of the present invention, there is provided apolypeptide of the formula:

HαDGTFTSDYSKYLDS-TtdsEC-CONH₂

In an embodiment of the present invention, there is provided apolypeptide of the formula:

(SEQ ID NO: 155) HαDGTFTSDYSKYLDSRRAQDFVQWLmNTKRNRNNIA-Ttds-EEEEEC-COOH,wherein Ttds is 1-amino-4,7,10-trioxa-13-tridecanamine succinimic acid.

In another embodiment of the present invention there is provided amethod for the treatment of a metabolic disease in a subject comprisingadministering to the subject a polypeptide as described above. Themetabolic disease may be selected from the group consisting of diabetes,metabolic syndrome, hyperglycemia, and obesity and may be administeredvia a route peripheral to the brain, such as an oral, mucosal, buccal,sublingual, nasal rectal, subcutaneous, transdermal intravenous,intramuscular or intraperitoneal route.

In yet another embodiment of the present invention, there is provided apharmaceutical composition comprising a polypeptide as described aboveand a pharmaceutically suitable carrier.

The present invention further relates to the use of the polypeptides ofthe present invention in the preparation of a medicament useful for thetreatment or prevention of metabolic disorders such as diabetes orobesity in a subject in need thereof by administering the polypeptidesand pharmaceutical compositions of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts activation of a mutant form of the human GLP-1 receptorby (a) native porcine OXM and (b) PEGylated OXM2 and loss of potency dueto preincubation of the peptides with DP-IV.

FIG. 2 shows incretin activity of porcine oxyntomodulin in the leanmouse intraperitoneal glucose tolerance test (IPGTT). % inhibition ofglucose excursion is indicated for each group. “ctrl”=vehicle-treatedsaline-challenged mice, “veh”=vehicle-treated dextrose-challenged mice.

FIGS. 3A, 3B, and 3C illustrate the efficacy of the polypeptides denotedby sequences OXM2 and OXM3 in reducing overnight food intake and bodyweight gain in lean mice. * p<0.05 relative to the vehicle group.

FIG. 4 depicts the effects of GLP-1 and OXM on glucose-stimulatedinsulin secretion (GSIS) in islets and MIN6 cells.

FIG. 5 shows the effects of GLP-1R deletion and receptor antagonism onOXM, GCG and GLP-1 mediated GSIS in islets.

FIG. 6 illustrates the effect of OXM on GSIS in glucagon receptor −/−islets.

FIG. 7 shows the effects of OXM and exendin-4 on blood glucose andinsulin levels during IPGTT in wild-type and GLP-1R−/− mice.

FIG. 8 shows the acute glucose-lowering effects of OXM99 in the leanmouse IPGTT.

FIG. 9 depicts the effect of OXM99 in reducing blood glucose in leanmice.

FIG. 10 illustrates the glucose-lowering effects of OXM117 in the leanmouse IPGTT.

FIG. 11 shows the duration of action of the OXM analogs OXM117 and OXM99compared with exendin-4.

FIG. 12 depicts the pharmacokinetics for OXM117 using subcutaneousdosing in the rat.

FIG. 13 shows the results of studies demonstrating that the OXM117peptide shows no glucagon-like activity in vitro.

FIG. 14 summarizes the in vitro activity data at the GLP1 and GCGreceptors in tabular form.

FIG. 15 shows in vivo activity of (mPEG)₂40 kDa conjugates on foodintake and body weight loss in the DIO mouse model.

FIG. 16 illustrates in vitro potency data for the C-terminal truncatedanalogs acting at the GLP1 and GCG receptors (OxmOH (SEQ ID NO:167),OxmNH₂ (SEQ ID NO:4), Oxm25 (SEQ ID NO:27), Oxm26 (SEQ ID NO:28), Oxm27(SEQ ID NO:29), Oxm90 (SEQ ID NO:94), Oxm91 (SEQ ID NO:95), Oxm92 (SEQID NO:96), Oxm93 (SEQ ID NO:97), GcgOH (SEQ ID NO:168)).

FIG. 17 presents in vitro potency data for select PEGylated OXM analogsacting at the GLP1 and GCG receptors.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to modified OXM derivatives. The OXM derivativesare developed by PEGylation or conjugation to other moieties or carrierproteins to improve stability and pharmacokinetics, and/or byincorporation of substitutions of amino acid residues to render thepeptides resistant to DP-IV cleavage. In addition, the stabilized OXMderivatives do not exhibit glucagon receptor agonist activity, and maythereby offer certain advantages in the treatment of hyperglycemia andobesity in diabetic or prediabetic subjects. For those subjects,up-regulation of glucagon receptor signaling should be avoided, since itmay result in elevated blood glucose levels.

Unless otherwise specified, the terms “polypeptide,” “protein,” and“peptide” is understood by the skilled artisan to also encompass variousmodified and/or stabilized forms. Such modified forms may be chemicallymodified forms, including, without limitation, PEGylated forms,palmitoylated forms, cholesterol-modified forms, etc. Modifications alsoinclude intra-molecular crosslinking and covalent attachment to variousmoieties such as lipids, flavin, biotin, polyethylene glycolderivatives, etc. In addition, modifications may also includecyclization, branching and cross-linking. Further, amino acids otherthan the conventional twenty amino acids encoded by genes may also beincluded in a polypeptide.

The Structures of the OXM Derivatives

The present invention provides modified OXM derivatives. In particular,the present invention relates to novel stabilized modified OXMpolypeptide derivatives of the formula:

(SEQ ID NO: 160) H_(x)X₁X₂GTFTSDYX₃X₄YLDX₅X₆X₆AX₇X₈FVX₇WLX₉X₁₀X₁₁KRNRNNX₁₂X₁₃X₁₄,

wherein H_(x) is selected from the group consisting of His;imidazole-lactic acid (ImiH); desamino-His (ΔNH₂-H); acetyl His;pyroglutamyl His (PyrH); N-methyl-His (Me-H); N,N-dimethyl-His (Me₂-H);Benzoyl His (Bz-H); Benzyl His (Bzl-H); and Phe;

X₁ is selected from the group consisting of Ser; Gly; Ala; Arg; Asn;Asp; Glu; Gln; His; Ile; Lys; Met; Phe; Pro; Thr; Trp; Tyr; Val; D-Ala;D-Ser; and α-aminoisobutyric acid;

X₂ is Gln, Asp, Glu, Pro, Leu or L-norleucine;

X₃ is Ser, Ala, Cys, Cys(mPEG), or Cys(cholesteryl);

X₄ is Lys, Cys, Cys(mPEG), or Cys(cholesteryl);

X₅ is Set or Ala;

X₆ is any amino acid;

X₇ is Gln, Cys, Cys(mPEG), or Cys(cholesteryl);

X₈ is Asp, Cys, Cys(mPEG), or Cys(cholesteryl);

X₉ is Met, Met(O), Val, norleucine, alanine, α-aminoisobutyric acid orO-methyl-homoserine;

X₁₀ is Asn, Cys, Cys(mPEG), or Cys(cholesteryl);

X₁₁ is Thr, Cys, Cys(mPEG), or Cys(cholesteryl);

X₁₂ is Ile, Cys, Cys(mPEG), or Cys(cholesteryl);

X₁₃ is Ala, Cys, Cys(mPEG), or Cys(cholesteryl); and

X₁₄ is amid; carboxylate, secondary amide, Ala, K(palmitoyl), Cys,Cys(mPEG), Cys(cholesteryl) or any linker to which mPEG or cholesterolis linked with a chemical bond.

Additionally, any one or two of X₃X₄, X₆-X₈, and X₁₀-X₁₄ may beCys(mPEG) or Cys(cholesteryl); Cys(mPEG)teine may also be C₁; C₂; C₃ orC₆, wherein C₁=Cys(mPEG)5 kDa, C₂=Cys(mPEG)20 kDa, C₃=Cys(mPEG)₂40 kDa,C₆=Cys(mPEG)₂60 kDa and each corresponds to a cysteine residue PEGylatedvia the side chain thiol with linear methoxyPEG (mPEG) or branched mPEG₂of the indicated MW.

The present invention further provides an OXM polypeptide of theformula:

(SEQ ID NO: 120) HαDGTFTSDYSKYLDSRRAQDFVQWLmNTKRNRNNIAC₃wherein C₃=Cys[(mPEG)₂40 kDa], each corresponding to an amidatedcysteine residue PEGylated via the side-chain thiol with a branched mPEG[(mPEG)₂] of the indicated MW; α is α-amino isobutyric acid (aib); andm=methionine sulfoxide [Met(O)].

In an embodiment of the present invention, there is provided apolypeptide comprising:

(SEQ ID NO: 162) HX₁QGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA

wherein X₁ is selected from the group consisting of Ser, Gly, Ala, Arg;Asn, Asp, Gln, Gln, His, Ile, Lys, Met, Phe, Pro, Thr, Trp, Tyr, Val,D-Ala, D-Ser, and α-aminoisobutyric acid.

The present invention further provides for a polypeptide comprising:

(SEQ ID NO: 163) HSX₂GTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA

X₂ is selected from the group consisting of Gln, Asp, Glu, Pro, Leu, andL-norleucine.

In another embodiment of the present invention, there is provided apolypeptide comprising:

(SEQ ID NO: 164) HSQGTFTSDYX₃X₄YLDSX₆X₆AX₇X₈FVX₇WLMX₁₀X₁₁KRNRNNX₁₂X₁₃X₁₄

wherein X₃ is Ser, Ala, Cys(mPEG), or Cys(cholesteryl);

X₄ is Lys, Cys(mPEG), or Cys(cholesteryl);

X₆ is Arg, Cys(mPEG), or Cys(cholesteryl);

X₇ is Gln, Cys(mPEG), or Cys(cholesteryl);

X₈ is Asp, Cys(mPEG), or Cys(cholesteryl);

X₁₀ is Asn, Cys(mPEG), or Cys(cholesteryl);

X₁₁ is Thr, Cys(mPEG), or Cys(cholesteryl);

X₁₂ is Ile, Cys(mPEG), or Cys(cholesteryl);

X₁₃ is Ala, Cys(mPEG), or Cys(cholesteryl); and X₁₄ is amide,carboxylate, secondary amide, Ala, K(palmitoyl), Cys(mPEG),Cys(cholesteryl), or any linker to which mPEG or cholesterol is linkedwith a chemical bond.

wherein one or two of X₃ X₄, X₆-X₈, and X₁₀-X₁₄ is Cys(mPEG) orCys(cholesteryl).

In an embodiment of the present invention, there is provided apolypeptide comprising:

(SEQ ID NO: 165) HαX₁₅GTFTSDYSKYLDSZZAX₁₆DFVQWLX₁₇NTX₁₈

wherein X₁₅ is D or Q;

Z is any amino acid;

X₁₆ is C8, Q or C;

X₁₇ is m or M;

X₁₈ is amidated k or K.

In another embodiment of the present invention, there is provided apolypeptide comprising:

(SEQ ID NO: 166) HαDGTFTSDYSKYDSZZAQDFVQWLmNTKRNRNNIAX₁₉,

wherein X₁₉ is C or C₈.

In yet another embodiment of the present invention, there is provided apolypeptide of the formula:

(SEQ ID NO: 152) HαDGTFTSDYSKYLDS-TtdsEC-CONH₂.

In an embodiment of the present invention, there is provided apolypeptide of the formula:

(SEQ ID NO: 155) HαDGTFTSDYSKYLDSRRAQDFVQWLmNTKRNRNNIA-Ttds-EEEEEC-COOH.

As used herein, the abbreviations of amino acid residues are shown asfollows:

Three-Letter One-Letter Amino Acids Abbreviations Abbreviations AlanineAla A Arginine Arg R Asparagine Asn N Aspartate Asp D Cysteine Cys CHistidine His H Isoleucine Ile I Glutamine Gln Q Glutamate Glu E GlycineGly G Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe FProline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine TyrY Valine Val V

Unless specifically designated otherwise, all the amino acid residuesare in the L-form.

In comparison to the wild-type OXM, the OXM derivatives of the presentinvention contain several amino acid substitutions, and/or can bePEGylated or otherwise modified (e.g. with cholesterol moieties).Analogs may be double conjugated, e.g., with to both cholesterol andPEG. Such OXM derivatives are resistant to cleavage and inactivation bydipeptidyl peptidase IV (DP-IV).

By “receptor agonist” is meant any endogenous or exogenous (drug)substance or compound that can interact with a receptor, for example,the GLP-1R or the glucagon receptor, and thereby initiate apharmacological or biochemical response characteristic of receptoractivation. Typically, the OXM derivatives of the instant invention arecharacterized by their affinity to the human GLP-1R and display an EC50for this receptor in the range of 0.1 pM to 1 μM. The OXM derivatives ofthe instant invention also are characterized by their affinity to theGcgR, displaying an EC50>1 μM.

The OXM derivatives of the present invention may be useful in thereduction of food intake and body weight and may mediateglucose-stimulated insulin secretion (GSIS) from pancreatic islets,thereby providing a treatment option for individuals afflicted with ametabolic disorder such as obesity, diabetes, metabolic syndrome X,hyperglycemia, impaired fasting glucose, and other prediabetic states.

TABLE 1 OXM Derivatives SEQ ID NO Peptides Sequences 1 OXM1HGQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIAA-COOH 2 OXM2HGQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIAC ₂-COOH 3 OXM3HGQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIAC ₃-COOH 4 OXM-NH₂HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA-CONH₂ 5 Ac-OXM-Ac-HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA-CONH₂ NH₂ 6 Ac-OXMAc-HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA-COOH 7 OXM4HαQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA-COOH 8 OXM5HVQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA-COOH 9 OXM6HaQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA-COOH 10 OXM7HsQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA-COOH 11 OXM8HSEGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA-COOH 12 OXM9HSDGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA-COOH 13 OXM10HSLGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA-COOH 14 OXM11HSnGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA-COOH 15 OXM12HGEGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA-COOH 16 OXM13FSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA-COOH 17 OXM14Pyr-HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA-CONH₂ 18 OXM15HSPGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA-COOH 19 OXM16 H₁SQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA-CONH₂ 20 OXM17Me-HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA-CONH₂ 21 OXM18 H₂SQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA-CONH₂ 22 OXM19 Me₂-HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA-CONH₂ 23 OXM20Bz-HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA-CONH₂ 24 OXM21Bzl-HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA-CONH₂ 25 OXM23HAEGTFTSDVSSYLEGQAAKEFIAWLMNTKRNRNNIA-CONH₂ 26 OXM24HSQGTFTSDYAKYLDARRAQDFVQWLMNTKRNRNNIA-CONH₂ 27 OXM25HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNI-CONH₂ 28 OXM26HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNN-CONH₂ 29 OXM27HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRN-CONH₂ 30 OXM28HαQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA-CONH₂ 31 OXM29HαQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIAC-CONH₂ 32 OXM30HαQGTFTSDYSKYLDSRRAQDFVQWLMCTKRNRNNIA-CONH₂ 33 OXM31HsQGTFTSDYSKYLDSRRAQDFVQWLmNTKRNRNNIA-COOH 34 OXM32HsQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA-CONH₂ 35 OXM33-36HsQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIAC-CONH₂ precursor 36 OXM33HsQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIAC ₁-CONH₂ 37 OXM34HsQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIAC ₂-CONH₂ 38 OXM35HsQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIAC ₃-CONH₂ 39 OXM36HsQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIAC ₄-CONH₂ 40 OXM37HAQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA-CONH₂ 41 OXM38HRQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA-CONH₂ 42 OXM39HNQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA-CONH₂ 43 OXM40HDQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA-CONH₂ 44 OXM41HEQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA-CONH₂ 45 OXM42HQQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA-CONH₂ 46 OXM43HHQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA-CONH₂ 47 OXM44HIQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA-CONH₂ 48 OXM45HLQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA-CONH₂ 49 OXM46HKQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA-CONH₂ 50 OXM47HMQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA-CONH₂ 51 OXM48HFQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA-CONH₂ 52 OXM49HPQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA-CONH₂ 53 OXM50HTQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA-CONH₂ 54 OXM51HWQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA-CONH₂ 55 OXM52HYQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA-CONH₂ 56 OXM53HsEGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA-CONH₂ 57 OXM54HsQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIAC ₅-CONH₂ 58 OXM55-59HsQGTFTSDYSKYLDSRRACDFVQWLMNTKRNRNNIA-CONH₂ precursor 59 OXM55HsQGTFTSDYSKYLDSRRAC ₅DFVQWLMNTKRNRNNIA-CONH₂ 60 OXM56HsQGTFTSDYSKYLDSRRAC ₁DFVQWLMNTKRNRNNIA-CONH₂ 61 OXM57HsQGTFTSDYSKYLDSRRAC ₂DFVQWLMNTKRNRNNIA-CONH₂ 62 OXM58HsQGTFTSDYSKYLDSRRAC ₃DFVQWLMNTKRNRNNIA-CONH₂ 63 OXM59HsQGTFTSDYSKYLDSRRAC ₄DFVQWLMNTKRNRNNIA-CONH₂ 64 OXM60HsQGTFTSDYSKYLDSRRAQDFVQWLnNTKRNRNNIA-CONH₂ 65 OXM61HsQGTFTSDYSKYLDSRRAQDFVQWLMC ₅TKRNRNNIA-CONH₂ 66 OXM62HsQGTFTSDYSKYLDSRRAQDFVQWLMC ₁TKRNRNNIA-CONH₂ 67 OXM63HsQGTFTSDYSKYLDSRRAQDFVQWLMC ₂TKRNRNNIA-CONH₂ 68 OXM64HsQGTFTSDYSKYLDSRRAQDFVQWLMC ₃TKRNRNNIA-CONH₂ 69 OXM65HsQGTFTSDYSKYLDSRRAQDFVQWLMC ₄TKRNRNNIA-CONH₂ 70 OXM66HαQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIAC ₅-CONH₂ 71 OXM67HαQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIAC ₁-CONH₂ 72 OXM68HαQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIAC ₂-CONH₂ 73 OXM69HαQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIAC ₃-CONH₂ 74 OXM70HαQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIAC ₄-CONH₂ 75 OXM71HsEGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIAC-CONH₂ 76 OXM72HsEGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIAC ₅-CONH₂ 77 OXM73HsEGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIAC ₁-CONH₂ 78 OXM74HsEGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIAC ₂-CONH₂ 79 OXM75HsEGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIAC ₃-CONH₂ 80 OXM76HsEGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIAC ₄-CONH₂ 81 OXM77HsQGTFTSDYSKYLDSRRAQCFVQWLMNTKRNRNNIA-CONH₂ 82 OXM78HsQGTFTSDYSKYLDSRRAQC ₅FVQWLMNTKRNRNNIA-CONH₂ 83 OXM79HsQGTFTSDYSKYLDSRRAQC ₁FVQWLMNTKRNRNNIA-CONH₂ 84 OXM80HsQGTFTSDYSKYLDSRRAQC ₂FVQWLMNTKRNRNNIA-CONH₂ 85 OXM81HsQGTFTSDYSKYLDSRRAQC ₃FVQWLMNTKRNRNNIA-CONH₂ 86 OXM82HsQGTFTSDYSKYLDSRRAQC ₄FVQWLMNTKRNRNNIA-CONH₂ 87 OXM83HsQGTFTSDYSKYLDSRRAQDFVCWLMNTKRNRNNIA-CONH₂ 88 OXM84HsQGTFTSDYSKYLDSRRAQDFVC ₅WLMNTKRNRNNIA-CONH₂ 89 OXM85HsQGTFTSDYSKYLDSRRAQDFVC ₁WLMNTKRNRNNIA-CONH₂ 90 OXM86HsQGTFTSDYSKYLDSRRAQDFVC ₂WLMNTKRNRNNIA-CONH₂ 91 OXM87HsQGTFTSDYSKYLDSRRAQDFVC ₃WLMNTKRNRNNIA-CONH₂ 92 OXM88HsQGTFTSDYSKYLDSRRAQDFVC ₄WLMNTKRNRNNIA-CONH₂ 93 OXM89HsQGTFTSDYSKYLDSRRAQDFVQWLVNTKRNRNNIA-CONH₂ 94 OXM90HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNR-CONH₂ 95 OXM91HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRN-CONH₂ 96 OXM92HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKR-CONH₂ 97 OXM93HSQGTFTSDYSKYLDSRRAQDFVQWLMNTK-CONH₂ 98 OXN94HαQGTFTSDYSKYLDSRRAQDFVQWLmNTKRNRNNIAC-CONH₂ 99 OXM95HsDGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIAC ₅-CONH₂ 100 OXM96HαEGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIAC ₅-CONH₂ 101 OXM97HαDGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIAC ₅-CONH₂ 102 OXM98HαQGTFTSDYSKYLDSRRAQDFVQWLmNTKRNRNNIAC ₅-CONH₂ 103 OXM99HαQGTFTSDYSKYLDSRRAQDFVQWLmNTKRNRNNIAC ₃-CONH₂ 104 OXM100HαQGTFTSDYSKYLDSRRAQDFVQWLmNTKRNRNNIAC ₆-CONH₂ 105 OXM101HαQGTFTSDYSKYLDSRRAQDFVQWLmNTKRNRNNIAC ₄-CONH₂ 106 OXM102HαQGTFTSDYSKYLDSRRAC ₅DFVQWLMNTKRNRNNIA-CONH₂ 107 OXM103HαQGTFTSDYSKYLDSRRAC ₃DFVQWLMNTKRNRNNIA-CONH₂ 108 OXM104HαQGTFTSDYSKYLDSRRAQC ₅FVQWLMNTKRNRNNIA-CONH₂ 109 OXM105HαQGTFTSDYSKYLDSRRAQC ₃FVQWLMNTKRNRNNIA-CONH₂ 110 OXM106HαQGTFTSDYSKYLDSRRAQDFVC ₅WLmNTKRNRNNIA-CONH₂ 111 OXM107HαQGTFTSDYSKYLDSRRAQDFVC ₃WLmNTKRNRNNIA-CONH₂ 112 OXM108HαQGTFTSDYSKYLDSRRAQDFVQWLMC ₅TKRNRNNIA-CONH₂ 113 OXM109HαQGTFTSDYSKYLDSRRAQDFVQWLMC ₃TKRNRNNIA-CONH₂ 114 OXM110HsQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIAK(palmitoyl)- CONH₂ 115 OXM111 H ₂αQGTFTSDYSKYLDSRRAQDFVQWLmNTKRNRNNIAC-CONH₂ 116 OXM112 H ₂αQGTFTSDYSKYLDSRRAQDFVQWLmNTKRNRNNIAC ₅-CONH₂ 117 OXM113 H ₂αQGTFTSDYSKYLDSRRAQDFVQWLmNTKRNRNNIAC ₃-CONH₂ 118 OXM114 H ₂αQGTFTSDYSKYLDSRRAQDFVQWLmNTKRNRNNIAC ₄-CONH₂ 119 OXM116HαDGTFTSDYSKYLDSRRAQDFVQWLmNTKRNRNNIAC-CONH₂ 120 OXM117/145/HαDGTFTSDYSKYLDSRRAQDFVQWLmNTKRNRNNIAC ₃-CONH₂ 146 121 OXM118HαDGTFTSDYSKYLDSRRAQDFVQWLmNTKRNRNNIAC ₄-CONH₂ 122 OXM119HαQGTFTSDYCKYLDSRRAQDFVQWLmNTKRNRNNIA-CONH₂ 123 OXM120 HαQGTFTSDYC₅KYLDSRRAQDFVQWLmNTKRNRNNIA-CONH₂ 124 OXM121 HαQGTFTSDYC₃KYLDSRRAQDFVQWLmNTKRNRNNIA-CONH₂ 125 OXM122HαQGTFTSDYSCYLDSRRAQDFVQWLmNTKRNRNNIA-CONH₂ 126 OXM123 HαQGTFTSDYSC₅YLDSRRAQDFVQWLmNTKRNRNNIA-CONH₂ 127 OXM124 HαQGTFTSDYSC₃YLDSRRAQDFVQWLmNTKRNRNNIA-CONH₂ 128 OXM125 HαDGTFTSDYSKYLDSRRAC₃DFVQWLmNTKRNRNNIA-CONH₂ 129 OXM126HαQGTFTSDYSKYLDSRRAQDCVQWLmNTKRNRNNIA-CONH₂ 130 OXM127HαQGTFTSDYSKYLDSRRAQDC ₄VQWLmNTKRNRNNIA-CONH₂ 131 OXM128 H ₂αDGTFTSDYSKYLDSRRACDFVQWLmNTKRNRNNIA-CONH₂ 132 OXM129 H ₂αDGTFTSDYSKYLDSRRAC ₃DFVQWLmNTKRNRNNIA-CONH₂ 133 OXM130HαDGTFTSDYSKYLDSRRAQDFVQWLMNTK-CONH₂ 134 OXM131HαDGTFTSDYSKYLDSRRAQDFVQWLMNTK-CONH₂ 135 OXM132HαQGTFTSDYSKYLDSRRACDFVQWLmNTKRNRNNIA-CONH₂ 136 OXM133HαQGTFTSDYSKYLDSRRAC ₅DFVQWLmNTKRNRNNIA-CONH₂ 137 OXM134HαQGTFTSDYSKYLDSRRAC ₃DFVQWLmNTKRNRNNIA-CONH₂ 138 OXM135HαQGTFTSDYSKYLDSRRACDFVQWLmNTK-CONH₂ 139 OXM136 HαQGTFTSDYSKYLDSRRAC₃DFVQWLmNTK-CONH₂ 140 OXM137 HαDGTFTSDYSKYLDSRRACDFVQWLmNTK-CONH₂ 141OXM138 HαDGTFTSDYSKYLDSRRAC ₃DFVQWLmNTK-CONH₂ 142 OXM139HαDGTFTSDYSKYLDSRRAQDFVQWLmNTKRNRNNIAC ₈-CONH₂ 143 OXM141HαDGTFTSDYSKYLDSRRAC ₃DFVQWLmNTKRNRNNIAC ₄-CONH₂ 144 OXM142HαQGTFTSDYSKYLDSRRAC ₃DFVQWLmNTKRNRNNIAC ₄-CONH₂ 145 OXM143HαQGTFTSDYSKYLDSRRAC ₈DFVQWLmNTK-CONH₂ 146 OXM144 HαDGTFTSDYSKYLDSRRAC₈DFVQWLmNTK-CONH₂ 147 OXM147 HαQGTFTSDYSKYLDSRRACDFVQWLMNTK-CONH₂ 148OXM148 HαQGTFTSDYSKYLDSRRAC ₈DFVQWLMNTK-CONH₂ 149 OXM149HαDGTFTSDYSKYLDSRRACDFVQWLmNTk-CONH₂ 150 OXM150 HαDGTFTSDYSKYLDSRRAC₈DFVQWLmNTk-CONH₂ 151 OXM151HαDGTFTSDYSKYLDSEAAQDFVQWLmNTKRNRNNIAC-CONH₂ 152 OXM152HαDGTFTSDYSKYLDS-TtdsEC-CONH₂ 153 OXM153HαDGTFTSDYSKYLDSEAAQDFVQWLmNTKRNRNNIAC ₈-CONH₂ 154 OXM154HαDGTFTSDYSKYLDSEAAQDFVQWLmNTKRNRNNIAC ₃-CONH₂ 155 OXM155HαDGTFTSDYSKYLDSRRAQDFVQWLmNTKRNRNNIA-Ttds-EEEEEC-COOH 156 OXM174HαQGTFTSDYSKYLDSRRAC₃DFVQWLANTKRNRNNIA-CONH₂ 157 OXM175HαQGTFTSDYSKYLDSRRAC₃DFVQWLVNTKRNRNNIA-CONH₂ 158 OXM176HαQGTFTSDYSKYLDSRRAC ₃DFVQWLαNTKRNRNNIA-CONH₂ 159 OXM199HαQGTFTSDYSKYLDSRRAC ₃DFVQWLXNTKRNRNNIA-CONH₂ α= α-aminoisobutyric acid(Aib); a = D-Ala; s = D-Ser; n = L-norleucine (Nle), X =O-methyl-homoserine; C₁ = Cys (mPEG) 5 kDa, C₂ = Cys (mPEG) 20 kDa, C₃ =Cys (mPEG)₂ 40 kDa, each corresponding to a cysteine residue PEGylatedvia the side-chain thiol with linear methoxyPEG (mPEG) or branched mPEG[(mPEG)₂] of the indicated MW; C₄ = Cys (Cholesteryl), corresponding toa cysteine residue linked to cholesterol via the side-chain thiol; C₅ =Cys(CH₂CONH₂), corresponding to a cysteine residue in which theside-chain thiol was reacted with iodoacetamide; C₆ = Cys (mPEG)₂60 kDa,each corresponding to a cysteine residue PEGylated via the side-chainthiol with linear methoxyPEG (mPEG) or branched mPEG₂mPEG [(mPEG)₂] ofthe indicated MW; H₁ = Imidazole-lactic acid (ImiH); H₂ = desamino-His(ΔNH₂—H)¹ Ac = Acetyl; Pyr = pyroglutamyl; Me-H = N-methyl-His; Me₂-H =N,N-dimethyl-His; Bz = Benzoyl (C7H5O); Bzl = Benzyl(C7H7); m =methionine sulfoxide. C₇ = (Cys)₂ (mPEG)2-40 kDa, each corresponding totwo cysteine residues PEGylated via the side chain thiol to the same onelinear methoxyPEG (mPEG) or one branched mPEG [(mPEG)₂], C₈ = Cys(N-ethylmaleimidyl); Ttds, 1-amino-4,7,10-trioxa-13-tridecanaminesuccinimic acid; k, D-Lysine.1.1. Amino Acid Substitutions and Modifications

Substitution at X₁ (position 2 of OXM) is designed to improve theresistance of the OXM derivatives to proteolysis by DP-W, which plays akey role in the degradation of many peptides, including OXM and GLP-1.It has been reported that substitution of Ser at position 2 with Gly inGLP-1 improves resistance to DP-IV cleavage (Lotte, B. K., J. Med.Chem., 47:4128-4134 (2004)). In spite of the high degree of sequencehomology between OXM and GLP-1, the Ser→Gly substitution at position 2was not found to confer a similar effect on the modified OXM. However,the substitution of Ser at position 2 with Val, Ile, Asp, Glu, Met, Trp,Asn, D-Ala, D-Ser, or α-aminoisobutyric acid rendered the correspondingOXM derivative more resistant to DP-IV than the wild-type OXM, asdiscussed infra in the Examples. Peptides with a substitution at X₁(position 2 of OXM) include: OXM4-7, 12, 23, 28-59, and the precursorsof OXM33-36 and OXM55-59.

The substitutions at X₂ (position 3 of OXM) are designed to create OXMderivatives that are selective agonists of GLP-1R with minimal or noactivation of GcgR. Such OXM derivatives may be advantageous whentreating obese diabetics. Peptides with a substitution at X₂ (position 3of OXM) include: OXM8-12, 15, 23, 53, 95, 96 and 97.

Similarly, the substitutions to Ala at X₃ and X₅ (positions 11 and 16)are designed to create OXM derivatives that are selective for GLP-1R andhave no activity against GcgR. One such example of the substitutions toAla at X₃ and X₅ (positions 11 and 16) is OXM24.

The substitutions to cysteine at any one or more of positions X₃ X₄,X₆-X₈, and X₁₀-X₁₄ allow for the PEGylation or cholesterylation of theOXM derivative at specific sites. Other substitutions or modificationsare known in the art and include those which physically adhere to thesurface of the active agent but do not chemically bond to or interactwith the active agent. Two or more such modifications can be employedand may be selected from known organic and inorganic pharmaceuticalexcipients, including various polymers, low molecular weight oligomers,natural products, and surfactants.

1.2. PEGylation and/or Cholesterylation

The invention contemplates the use of multi-functional polymerderivatives, as exemplified by bifunctional and multi-arm N-maleimidylPEG derivatives. A wide variety of polyethylene glycol (PEG) species maybe used for PEGylation of the novel OXM derivatives of the presentinvention. Substantially any suitable reactive PEG reagent can be usedand suitable species include, but are not limited to, those which areavailable for sale in the Drug Delivery Systems catalog of NOFCorporation (Yebisu Garden Place Tower, 20-3 Ebisu 4-chome, Shibuya-ku,Tokyo 150-6019) and, for exemplary purposes, of the MolecularEngineering catalog of Nelctar Therapeutics (490 Discovery Drive,Huntsville, Ala. 35806). By way of example and not limitation, thefollowing PEG reagents are often preferred in various embodiments:multi-Arm PEG, mPEG(MAL)₂, mPEG2(MAL), any of the SUNBRIGHT activatedPEGs (including but not limited to carboxyl-PEGs, p-NP-PEGs,Tresyl-PEGs, aldehyde PEGs, acetal-PEGs, amino-PEGs, thiol-PEGs,maleimido-PEGs, hydroxyl-PEG-amine, amino-PEG-COOH,hydroxyl-PEG-aldehyde, carboxylic anhydride type-PEG, functionalizedPEG-phospholipid), and other similar and/or suitable reactive PEGs asselected by those skilled in the art for their particular applicationand usage.

The novel OXM derivative peptides of the present invention can alsocontain two PEG moieties that are covalently attached via a carbamate oran amide linkage to a spacer moiety, wherein the spacer moiety iscovalently bonded to the tertiary amide linker of the peptide. Each ofthe two PEG moieties used in such embodiments of the present inventionmay be linear and may be linked together at a single point ofattachment. In one embodiment of the invention, each PEG moiety has amolecular weight of about 10 kilodaltons (10K) to about 60K (the term“about” indicating that in preparations of PEG, some molecules willweigh more, and some less, than the stated molecular weight). Each ofthe two PEG moieties may have a molecular weight of about 20K to about40K. One skilled in the art will be able to select the desired polymersize based on such considerations as the desired dosage; circulationtime; resistance to proteolysis; effects, if any, on biologicalactivity; ease in handling; degree or lack of antigenicity; and otherknown effects of PEG on a therapeutic peptide.

In an embodiment of the present invention, the polymer backbone of theN-maleimidyl polymer derivative is a poly(alkylene glycol), copolymerthereof, terpolymer thereof, or mixture thereof. Examples includepoly(ethylene glycol), poly(propylene glycol), and copolymers ofethylene glycol and propylene glycol. As explained in greater detailbelow, more preferred embodiments of the invention utilize PEG polymers,such as bifunctional PEG, multiarmed PEG, forked PEG, branched PEG,pendent PEG, and PEG with degradable linkages therein. However, itshould be understood that other related polymers are also suitable foruse in the practice of this invention and that the use of the term PEGor poly(ethylene glycol) is intended to be inclusive and not exclusivein this respect. The term PEG includes poly(ethylene glycol) in any ofits forms, including bifunctional PEG, multiarmed PEG, forked PEG,branched PEG, pendent PEG (i.e. PEG or related polymers having one ormore functional groups pendent to the polymer backbone), or PEG withdegradable linkages therein.

The polymer backbone can be linear or branched. PEG is commonly used inbranched forms that can be prepared by addition of ethylene oxide tovarious polyols, such as glycerol, glycerol oligomers, pentaerythritoland sorbitol. The central branch moiety can also be derived from severalamino acids, such as lysine. The branched poly(ethylene glycol) can berepresented in general form as R(-PEG-OH)_(m) in which R is derived froma core moiety, such as glycerol, glycerol oligomers, or pentaerythritol,and m represents the number of arms. Multi-armed PEG molecules, such asthose described in U.S. Pat. No. 5,932,462, which is incorporated byreference herein in its entirety, can also be used as the polymerbackbone.

Those of ordinary skill in the art will recognize that the foregoinglist for substantially water soluble and non-peptidic polymer backbonesis by no means exhaustive and is merely illustrative, and that allpolymeric materials having the qualities described above arecontemplated.

The sites of PEGylation on the OXM derivatives of the present inventionare chosen taking into account the structure of OXM and its interactionswith glucagon and GLP-1 receptors. Hence, the PEGylation is preferablysite-specific. PEGylation at the thiol side-chain of cysteine has beenwidely reported (see, e.g., Caliceti & Veronese, 2003). If there is noCys residue in the peptide, it can be introduced through substitution.The OXM derivatives of the present invention may be PEGylated throughthe side chains of cysteine. The OXM derivatives may containCys(mPEG)teine. The mPEG in Cys(mPEG)teine can have various molecularweights. The range of the molecular weight is preferably 5 kDa to 200kDa, more 5 kDa to 100 kDa, and further preferably 20 kDa to 60 kDA. ThemPEG can be linear or branched. For instance, the Cys(mPEG)teine ofpresent invention may be C₁, C₂, C₃ or C₆. As exemplified herein, C₁ isCys(mPEG)teine with a linear mPEG with a molecular weight of 5 kDa(Cys(mPEG)₅ kDa) (e.g., MPEG-MAL-5000, NEKTAR 2F2MOH01); C₂ isCys(mPEG)teine with a linear mPEG with a molecular weight of 20 kDa(Cys(mPEG)20 kDa) (e.g., MPEG-MAL-20K, NEKTAR 2F2M0P01); C₃ isCys(mPEG)teine with a branched mPEG with a molecular weight of 40 kDa(Cys(mPEG)₂40 kDa) (e.g., MPEG2-MAL-40K, NEKTAR 2D3Y0T01 or Y Shape PEGMaleimide, MW40K (JenKem Technology, item number Y-MAL-40K or SUNBRIGHTGL2-400MA Maleimide, (NOF Corporation) and C₆ is Cys(mPEG)teine with abranched mPEG with a molecular weight of 60 kDa (Cys(mPEG)₂60 kDa)(e.g., MPEG2-MAL-60K, NEKTAR 2D3Y0V01).

Alternatively, the cysteine residues in the OXM derivatives can also bederivatized with cholesterol via the side-chain thiol. Examples ofcholesteryl OXM derivatives include: OXM36, OXM59, OXM65, OXM70, OXM76,OXM82, OXM88, and OXM101.

1.3. Other Modifications

The N-terminal Histidine H_(x) can be mutated with derivatives from thegroup consisting of His, H₁=Imidazole-lactic acid (ImiH); desamino-His(ΔNH₂—H), acetyl His, pyroglutamyl His(PyrH, N-methyl-His (Me-H),N,N-dimethyl-His (Me₂-H); Benzoyl His (Bz-H), Benzyl His (Bzl-H) andPhe. Acetylation and other modification and N-terminal capping groups atthe N-terminus may stabilize OXM against DP-IV cleavage, while theamidation of C-terminus may prevent potential degradation in vivo bycarboxypeptidases. OXM derivatives with N-terminal modifications includeOXM14, and OXM16-22.

As illustrated in the examples, equimolar doses of OXM2 and OXM3 wereeffective in reducing overnight body weight gain in mice fed ad libitum,whereas similar doses of OXM1 and wild type or native (wt) Oxm were notefficacious in this model. OXM3 had the highest in vivo efficacy indose-dependently reducing overnight body weight gain, likely reflectingits higher potency against GLP-1R compared to OXM2, which has a bulkierPEG moiety that likely interferes with receptor binding. For thePEGylated OXM derivatives, increased in vivo efficacy relative to wt OXMsuggests that some stabilization against proteolysis, and for renalclearance, is induced by PEGylation alone, since both OXM2 and OXM3 aresignificantly less potent than native OXM against GLP-1R in vitro.

Additionally, a blood component may be utilized to stabilize thepeptide. Preferred blood components comprise proteins such asimmunoglobulins, serum albumin, ferritin, steroid binding proteins,transferrin, thyroxin binding protein, alpha.-2-macroglobulin,haptoglobin and the like.

2. Synthesis of the OXM Derivatives

2.1. The Synthesis of Peptides

The following general procedure was used to synthesize some of the OXMderivatives. Solid phase peptide synthesis was performed using Fmocchemistry under batch or continuous flow conditions (see, for example,Pennington and Dunn, Peptide Synthesis Protocols (1994), vol. 35) usingPEG-polystyrene resins. Peptides were cleaved from the resin anddeprotected using trifluoroacetic acid (TFA), and cation scavengers suchas phenol triisopropylsilane, and water. Peptides were precipitated withcold methyl-t-butyl ether and the precipitated peptide was washed twicewith cold ether prior to lyophilization. Peptide identity was confirmedby reversed-phase HPLC on a C4 column using water/acetonitrile with 0.1%TFA as typical mobile phases, and by electrospray mass spectrometry.Peptides were purified to ≧95% by reverse phase HPLC.

2.2 PEGylation of Peptides

Peptides are first synthesized and are then PEGylated at the thiolside-chain of cysteine. The following general procedure was used forPEGylation of peptides.

PEGylation reactions were run between a thiolated peptide precursor anda maleimide-mPEG to form a thioether bond. The reaction was run at a pH7.3 and the maleimide-mPEG amount ranged from 0.5 to 10-fold molarexcess with respect to the thiolated peptide. The PEGylated OXM peptidewas then isolated using reverse-phase HPLC or ion-exchangechromatography followed by size exclusion chromatography. Finally,PEGylated peptides were characterized using analytical RP-HPLC, andMALDI tof mass spectrometry.

3. Implications of OXM-Based Therapy

OXM-based therapy has the potential to favorably impact both obesity anddiabetes. Weight loss efficacy and reduction in food intake uponperipheral administration of OXM has been well validated in humans.Studies by the present inventors have shown that peripherallyadministered porcine OXM is sufficient to reduce short term food intakeand overnight body weight gain in mice. Although the incretin(antihyperglycemic) activity of OXM has not been well investigated todate, it has been demonstrated for the first time that the glucoselowering activity of OXM is comparable to that of GLP-1 in a mouseintraperitoneal glucose tolerance test (IPGTT). Like GLP-1, OXM inducesrobust glucose stimulated insulin secretion (GSIS) from static isolatedmurine islets and perfused rat pancreata (Jarrousse et al.,Endocrinology, 115:102-105 (1984)), suggesting a low risk ofhypoglycemia compared to conventional insulin secretagogues. In rats,negligible effects of OXM on gastric emptying have been reported (Dakinet al., Endocrinology, 145:2687-2695 (2004)). In mice, OXM reducesgastric emptying by ˜25% at a maximally efficacious dose for glucoselowering, which is less than that produced by a maximally efficaciousdose of the GLP-1 receptor agonist exendin 4 (47% reduction).Potentially benign effects of OXM on gastric emptying in humans maytherefore play a role in the enhanced tolerability of this peptidehormone compared to current GLP-1 mimetics.

It is suggested that the polypeptides of the present invention may beuseful for the treatment of obesity and/or diabetes. Secondaryindications are metabolic syndrome, hyperglycemia, impaired fastingglucose, and other prediabetic states. Alternate indications for thepolypeptides of the present invention include any and all indicationsfor GLP-1 such as irritable bowel syndrome and other absorptive diseasesof the gut, ischemia, stroke, and neurological disorders includinganxiety, impaired cognition, and Alzheimer's disease.

The peptidyl nature of OXM precludes oral therapy with the nativehormone. By contrast, the OXM derivatives presented herein may beadministered as a pharmaceutical composition comprising one of thepolypeptides of the present invention in combination with apharmaceutically acceptable carrier which is suitable for administrationby a variety of routes, including but not limited to oral, intranasal,sublingual, intraduodenal, subcutaneous, buccal, intracolonic, rectal,vaginal, mucosal, pulmonary, transdermal, intradermal, parenteral,intravenous, intramuscular and intraocular at a dosage range of 0.001mg/kg to 10 mg/kg, more preferably from 1 μg/kg to 200 mg/kg with adosing frequency ranging from twice daily to once per week or longer.The peptide pharmaceutical compositions can be administered in a varietyof unit dosage forms depending upon the method of administration.Suitable unit dosage forms, include, but are not limited to powders,tablets, pills, capsules, lozenges, suppositories, patches, nasalsprays, injectables, implantable sustained-release formulations, lipidcomplexes, etc. The peptides are typically combined with apharmaceutically acceptable carrier or excipient which can contain oneor more physiologically acceptable compound(s) that may act to stabilizethe composition or to increase or decrease the absorption of the activeagent(s). Physiologically acceptable compounds can include, for example,carbohydrates, such as glucose, sucrose, or dextran, antioxidants, suchas ascorbic acid or glutathione, chelating agents, low molecular weightproteins, protection and uptake enhancers such as lipids, compositionsthat reduce the clearance or hydrolysis of the active agents, orexcipients or other stabilizers and/or buffers. Other physiologicallyacceptable compounds include wetting agents, emulsifying agents,dispersing agents or preservatives that are particularly useful forpreventing the growth or action of microorganisms. Various preservativesare well known and include, for example, phenol and ascorbic acid. Oneskilled in the art would appreciate that the choice of pharmaceuticallyacceptable carrier(s), including a physiologically acceptable compounddepends, for example, on the route of administration of the activeagent(s) and on the particular physio-chemical characteristics of theactive agent(s).

The peptides can be administered in the native form or, if desired, inthe form of salts provided the salts are pharmaceutically acceptable.Salts of the active agents may be prepared using standard proceduresknown to those skilled in the art of synthetic organic chemistry.Polypeptides of the present invention, as detailed in the Examples, wereprepared as acetate salts.

An OXM polypeptide of the instant invention can be used in combinationwith other agents used in the treatment or prevention of diseasesimplicating the GLP1-R. Specific compounds of use in combination with apolypeptide of the present invention include: simvastatin, mevastatin,ezetimibe, atorvastatin, sitagliptin, metformin, sibutramine, orlistat,Qnexa, topiramate, naltrexone, bupriopion, phentermine, and losartan,losartan with hydrochlorothiazide. Specific CB1 antagonists/inverseagonists of use in combination with a polypeptide of the presentinvention include: those described in WO03/077847, including:N[3-(4-chlorophenyl)-2(S)-phenyl-[(S)-methylpropyl]-2-(4-trifluoromethyl-2-pyrimidyloxy)-2-methylpropanamide,N-[3-(4-chlorophenyl)-2-(3-cyanophenyl)-1-methylpropyl]-2-(5-trifluoromethyl-2-pyridyloxy)-2-methylpropanamide,N-[3-(4-chlorophenyl)-2-(5-chloro-3-pyridyl)-1-methylpropyl]-2-(5-trifluoromethyl-2-pyridyloxy)-2-methylpropanamide,and pharmaceutically acceptable salts thereof; as well as those inWO05/000809, which includes the following:3-{1-[bis(4-chlorophenyl)methyl]azetidin-3-ylidene}-3-(3,5-difluorophenyl)-2,2-dimethylpropanenitrile,1-{1-[1-(4-chlorophenyl)pentyl]azetidin-3-yl}-1-(3,5-difluorophenyl)-2-methylpropan-2-ol.3-((S)-(4-chlorophenyl){3-[(1S)-1-(3,5-difluorophenyl)-2-hydroxy-2-methylpropyl]azetidin-1-yl}methyl)benzonitrile,3-((S)-(4-chlorophenyl){3-[(1S)-1-(3,5-difluorophenyl)-2-fluoro-2-methylpropyl]azetidin-1-yl}methyl)benzonitrile,3-((4-chlorophenyl){3-[1-(3,5-difluorophenyl)-2,2-dimethylpropyl]azetidin-1-yl}methyl)benzonitrile,3-((1S)-1-{1-[(S)-(3-cyanophenyl)(4-cyanophenyl)methyl]azetidin-3-yl}-2-fluoro-2-methylpropyl)-5-fluorobenzonitrile,3-[(S)-(4-chlorophenyl)(3-{(1S)-2-fluoro-1-[3-fluoro-5-(4H-1,2,4-triazol-4-yl)phenyl]-2-methylpropyl}azetidin-1-yl)methyl]benzonitrile,and5-((4-chlorophenyl){3-[(1S)-1-(3,5-difluorophenyl)-2-fluoro-2-methylpropyl]azetidin-1-yl}methyl)thiophene-3-carbonitrile,and pharmaceutically acceptable salts thereof; as well as:3-[(S)-(4-chlorophenyl)(3-{(1S)-2-fluoro-1-[3-fluoro-5-(5-oxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)phenyl]-2-methylpropyl}azetidin-1-yl)methyl]benionitrile,3-[(S)-(4-chlorophenyl)(3-{(1S)-2-fluoro-1-[3-fluoro-5-(1,3,4-oxadiazol-2-yl)phenyl]-2-methylpropyl}azetidin-1-yl)methyl]benzonitrile,3-[(S)-(3-{(1S)-1-[3-(5-amino-1,3,4-oxadiazol-2-yl)-5-fluorophenyl]-2-fluoro-2-methylpropyl}azetidin-1-yl)(4-chlorophenyl)methyl]benzonitrile,3-[(S)-(4-cyanophenyl)(3-{(1S)-2-fluoro-1-[3-fluoro-5-(5-oxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)phenyl]-2-methylpropyl}azetidin-1-yl)methyl]benzonitrile,3-[(S)-(3-{(1S)-1-[3-(5-amino-1,3,4-oxadiazol-2-yl)-5-fluorophenyl]-2-fluoro-2-methylpropyl}azetidin-1-yl)(4-cyanophenyl)methyl]benzonitrile,3-[(S)-(4-cyanophenyl)(3-{(1S)-2-fluoro-1-[3-fluoro-5-(1,3,4-oxadiazol-2-yl)phenyl]-2-methylpropyl}azetidin-1-yl)methyl]benzonitrile,3-[(S)-(4-chlorophenyl)(3-{(1S)-2-fluoro-1-[3-fluoro-5-(1,2,4-oxadiazol-3-yl)phenyl]-2-methylpropyl}azetidin-1-yl)methyl]benzonitrile,3-[(1S)-1-(1-{(S)-(4-cyanophenyl)[3-(1,2,4-oxadiazol-3-yl)phenyl]-methyl}azetidin-3-yl)-2-fluoro-2-methylpropyl-]-5-fluorobenzonitrile,5-(3-{1-[1-(diphenylmethyl)azetidin-3-yl]-2-fluoro-2-methylpropyl}-5-fluorophenyl)-1H-tetrazole,5-(3-{1-[1-(diphenylmethyl)azetidin-3-yl]-2-fluoro-2-methylpropyl}-5-fluorophenyl)-1-methyl-1H-tetrazole,5-(3-{1-[1-(diphenylmethyl)azetidin-3-yl]-2-fluoro-2-methylpropyl}-5-fluorophenyl)-2-methyl-2H-tetrazole,3-[(4-chlorophenyl)(3-{2-fluoro-1-[3-fluoro-5-(2-methyl-2H-tetrazol-5-yl)phenyl]-2-methylpropyl}azetidin-1-yl)methyl]benzonitrile,3-[(4-chlorophenyl)(3-{2-fluoro-1-[3-fluoro-5-(1-methyl-1H-tetrazol-5-yl)phenyl]-2-methylpropyl}azetidin-1-yl)methyl]benzonitrile,3-[(4-cyanophenyl)(3-{2-fluoro-1-[3-fluoro-5-(1-methyl-1H-tetrazol-5-yl)phenyl]-2-methylpropyl}azetidin-1-yl)methyl]benzonitrile,3-[(4-cyanophenyl)(3-{2-fluoro-1-[3-fluoro-5-(2-methyl-2H-tetrazol-5-yl)phenyl]-2-methylpropyl}azetidin-1-yl)methyl]benzonitrile,5-{3-[(S)-{3-[(1S)-1-(3-bromo-5-fluorophenyl)-2-fluoro-2-methylpropyl]azetidin-1-yl}(4-chlorophenyl)methyl]phenyl}-1,3,4-oxadiazol-2(3H)-one,3-[(1S)-1-(1-{(S)-(4-chlorophenyl)[3-(5-oxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)phenyl]methyl}azetidin-3-yl)-2-fluoro-2-methylpropyl]-5-fluorobenzonitrile,3-[(1S)-1-(1-{(S)-(4-cyanophenyl)[3-(5-oxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)phenyl]methyl}azetidin-3-yl)-2-fluoro-2-methylpropyl]-5-fluorobenzonitrile,3-[(1S)-1-(1-{(S)-(4-cyanophenyl)[3-(1,3,4-oxadiazol-2-yl)phenyl]methyl}azetidin-3-yl)-2-fluoro-2-methylpropyl]-5-fluorobenzonitrile,3-[(1S)-1-(1-{(S)-(4-chlorophenyl)[3-(1,3,4-oxadiazol-2-yl)phenyl]methyl}azetidin-3-yl)-2-fluoro-2-methylpropyl]-5-fluorobenzonitrile,3-((1S)-1-{1-[(S)-[3-(5-amino-1,3,4-oxadiazol-2-yl)phenyl](4-chlorophenyl)methyl]azetidin-3-yl}-2-fluoro-2-methylpropyl)-5-fluorobenzonitrile,3-((1S)-1-{1-[(S)-[3-(5-amino-1,3,4-oxadiazol-2-yl)phenyl](4-cyanophenyl)methyl]azetidin-3-yl}-2-fluoro-2-methylpropyl)-5-fluorobenzonitrile,3-[(1S)-1-(1-{(S)-(4-cyanophenyl)[3-(1,2,4-oxadiazol-3-yl)phenyl]methyl}azetidin-3-yl)-2-fluoro-2-methylpropyl]-5-fluorobenzonitrile,3-[(1S)-1-(1-{(S)-(4-chlorophenyl)[3-(1,2,4-oxadiazol-3-yl)phenyl]methyl}azetidin-3-yl)-2-fluoro-2-methylpropyl]-5-fluorobenzonitrile,5-[3-((S)-(4-chlorophenyl){3-[(1S)-1-(3,5-difluorophenyl)-2-fluoro-2-methylpropyl]azetidin-1-yl]methyl)phenyl}-1,3,4-oxadiazol-2(1H)-one,5-[3-((S)-(4-chlorophenyl){3-[(1S)-1-(3,5-difluorophenyl)-2-fluoro-2-methylpropyl]azetidin-1-yl}methyl)phenyl]-1,3,4-oxadiazol-2(3H)-one,4-{(8)-{3-[(15)-1-(3,5-difluorophenyl)-2-fluoro-2-methylpropyl]azetidin-1-yl}[3-(5-oxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)phenyl]methyl}-benzonitrile,and pharmaceutically acceptable salts thereof.

Specific NPY5 antagonists of use in combination with a polypeptide ofthe present invention include:3-oxo-N-(5-phenyl-2-pyrazinyl)-spiro[isobenzofuran-1(3H),4′-piperidine]-1′-carboxamide,3-oxo-N-(7-trifluoromethylpyrido[3,2-b]pyridin-2-yl)spiro-[isobenzofuran-1(3H),4′-piperidine]-1′-carboxamide,N-[5-(3-fluorophenyl)-2-pyrimidinyl]-3-oxospiro-[isobenzofuran-1(3H),4′-piperidine]-1′-carboxamide,trans-3′-oxo-N-(5-phenyl-2-pyrimidinyl)spiro[cyclohexane-1,1′(3′H)-isobenzofuran]-4-carboxamide,trans-3′-oxo-N-[1-(3-quinolyl)-4-imidazolyl]spiro[cyclohexane-1,1′(3′H)-isobenzofuran]-4-carboxamide,trans-3-oxo-N-(5-phenyl-2-pyrazinyl)spiro[4-azaiso-benzofuran-1(3H),1′-cyclohexane]-4′-carboxamide,trans-N-[5-(3-fluorophenyl)-2-pyrimidinyl]-3-oxospiro[5-azaisobenzofuran-1(3H),1′-cyclohexane]-4′-carboxamide,trans-N-[5-(2-fluorophenyl)-2-pyrimidinyl]-3-oxospiro[5-azaisobenzofuran-1(3H),1′-cyclohexane]-4′-carboxamide,trans-N-[1-(3,5-difluorophenyl)-4-imidazolyl]-3-oxospiro[7-azaisobenzofuran-1(3H),1′-cyclohexane]-4′-carboxamide,trans-3-oxo-N-(1-phenyl-4-pyrazolyl)spiro[4-azaisobenzofuran-1(3H),1′-cyclohexane]-4′-carboxamide,trans-N-[1-(2-fluorophenyl)-3-pyrazolyl]-3-oxospiro[6-azaisobenzofuran-1(3H),1′-cyclohexane]-4′-carboxamide,trans-3-oxo-N-(1-phenyl-3-pyrazolyl)spiro[6-aisobenzofuran-1(3H),1′-cyclohexane]-4′-carboxamide,trans-3-oxo-N-(2-phenyl-1,2,3-triazol-4-yl)spiro[6-azaisobenzofuran-1(3H),1′-cyclohexane]-4′-carboxamide, and pharmaceutically acceptable saltsand esters thereof.

Specific ACC-1/2 inhibitors of use in combination with a polypeptide ofthe present invention include:1′-[(4,8-dimethoxyquinolin-2-yl)carbonyl]-6-(1H-tetrazol-5-yl)spiro[chroman-2,4′-piperidin]-4-one,(5-{1′-[4,8-dimethoxyquinolin-2-yl)carbonyl]-4-oxospiro[chroman-2,4′-piperidin]-6-yl}-2H-tetrazol-2-yl)methylpivalate,5-{1′-[(8-cyclopropyl-4-methoxyquinolin-2-yl)carbonyl]-4-oxospiro[chroman-2,4′-piperidin]-6-yl}nicotinicacid,1′-(8-methoxy-4-morpholin-4-yl-2-naphthoyl)-6-(1H-tetrazol-5-yl)spiro[chroman-2,4′-piperidin]-4-one,and1′-[(4-ethoxy-8-ethylquinolin-2-yl)carbonyl]-6-(1H-tetrazol-5-yl)spiro[chroman-2,4′-piperidin]-4-one,and pharmaceutically acceptable salts thereof.

Specific MCH1R antagonist compounds of use in combination with apolypeptide of the present invention include:1-{4-[(1-ethylazetidin-3-yl)oxy]phenyl}-4-[(4-fluorobenzyl)oxy]pyridin-2(1H)-one,4-[(4-fluorobenzyl)oxy]-1-{4-[(1-isopropylazetidin-3-yl)oxy]phenyl}pyridin-2(1H)-one,1-[4-(azetidin-3-yloxy)phenyl]-4-[(5-chloropyridin-2-yl)methoxy]pyridin-2(1H)-one,4-[(5-chloropyridin-2-yl)methoxy]-1-{4-[(1-ethylazetidin-3-yl)oxy]phenyl}pyridin-2(1H)-one,4-[(5-chloropyridin-2-yl)methoxy]-1-{4-[(1-propylazetidin-3-yl)oxy]phenyl}pyridin-2(1H)-one,and4-[(5-chloropyridin-2-yl)methoxy]-1-(4-{[(25)-1-ethylazetidin-2-yl]methoxy}phenyl)pyridin-2(1H)-one,or a pharmaceutically acceptable salt thereof.

Specific DP-IV inhibitors of use in combination with a polypeptide ofthe present invention are selected from7-[(3R)-3-amino-4-(2,4,5-trifluorophenyl)butanoyl]-3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,2,4-triazolo[4,3-a]pyrazine.In particular, the compound of formula I is favorably combined with7-[(3R)-3-amino-4-(2,4,5-trifluorophenyl)butanoyl]-3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,2,4-triazolo[4,3-a]pyrazine,and pharmaceutically acceptable salts thereof.

Additionally, other peptide analogs and mimetics of the incretin hormoneglucagon-like peptide 1(GLP-1), may also be of use in combination with apolypeptide of the present invention.

Other features and advantages of the present invention are apparent fromthe additional descriptions provided herein including the differentexamples. The provided examples illustrate different components andmethodology useful in practicing the present invention. The examples donot limit the claimed invention. Based on the present disclosure theskilled artisan can identify and employ other components and methodologyuseful for practicing the present invention.

EXAMPLES Example 1 Synthesis of Oxyntomodulin (OXM) Analogs

The peptide OXM analogs (see Table 1) were synthesized by solid phaseusing Fmoc/tBu chemistry on a peptide multisynthesizer APEX 396(Advanced Chemtech) using a 40-well reaction block. Each peptide wassynthesized in a single well. For peptide amides 0.1 g of a resinFmoc-Linker AM-Champion, 1% cross-linked (Biosearch Technologies, Inc.)and a PEG-PS based resin derivatized with a modified Rink linkerp-[(R,S)-α-[9H-Fluoren-9-yl-methoxyformamido]-2,4-dimethoxybenzyl]-phenoxyaceticacid (Rink, H., 1987, Tetrahedron Lett. 28:3787-3789; Bernatowicz, M. S.et al., 1989, Tetrahedron Lett. 30:4645-4667) was used. For peptideacids, 0.1 g of Champion resin, 1% cross-linked (Biosearch Technologies,Inc.) was used, which was previously derivatized with ahydroxymethylphenoxymethyl handle. All the amino acids were dissolved ata 0.5 M concentration in a solution of 0.5M HOBt (Hydroxybenzotriazole)in DMF. The acylation reactions were performed for 60 min with 6-foldexcess of activated amino acid over the resin free amino groups. Theamino acids were activated with equimolar amounts of HBTU(2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate) and a 2-fold molar excess of DIEA(N,N-diisopropylethylamine).

Alternatively, the peptides were synthesized by solid phase usingFmoc/t-Bu chemistry on a Pioneer Peptide Synthesizer (AppliedBiosystems). In this case, all the acylation reactions were performedfor 60 minutes with a 4-fold excess of activated amino acid over theresin free amino groups following the end of peptide assembly on thesynthesizer. The side chain protecting groups were: tert-butyl for Asp,Glu, Ser, Thr and Tyr; trityl for Asn, Cys, Gln and His;tert-butoxy-carbonyl for Lys, Trp; and,2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl for Arg. For the OXM2and OXM3 peptides, the acetylation reaction was performed at the end ofthe peptide assembly by reaction with a 10-fold excess of aceticanhydride in DMF.

For OXM14, L-pyroglutamic acid was acylated by reaction with equimolaramounts of DIPC (diisopropylcarbodiimide) and HOBt(N-hydroxybenzotriazole) with a 4-fold excess of activated acylant overthe resin free amino groups.

For OXM16, imidazole-lactic acid (Imi-H) was acylated by reaction withequimolar amounts of PyBOP(Benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphoniumhexafluorophosphate), HOBt and a 2-fold molar excess of DIEA(N,N-diisopropylethylamine) with a 4-fold excess of activated acylantover the resin free amino groups.

For OXM17, N-methyl-His (Me-H) was acylated by reaction with equimolaramounts of HBTU (2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate) and a 2-fold molar excess of DMA. The acylationreaction was performed for 180 min with a 3-fold excess of activatedacylant over the resin free amino groups.

For OXM18, desamino-His (ΔNH₂—H) was acylated by reaction with equimolaramounts of HBTU and a 2-fold molar excess of DIEA. The acylationreaction was performed for 180 min with a 3-fold excess of activatedacylant over the resin free amino groups.

For OXM19, N,N-dimethyl-His (Me₂-H) was acylated by reaction withequimolar amounts of HBTU and a 2-fold molar excess of DMA. Theacylation reaction was performed overnight with a 3-fold excess ofactivated acylant over the resin free amino groups.

For OXM20, benzoyl-His (Bz-H) was acylated by reaction with equimolaramounts of HBTU and a 2-fold molar excess of DIEA. The acylationreaction was performed for 240 min with a 3-fold excess of activatedacylant over the resin free amino groups.

For OXM21, Benzyl-His (Bzl-H) was acylated by reaction with equimolaramounts of HBTU and a 2-fold molar excess of DIEA. The acylationreaction was performed overnight with a 3-fold excess of activatedacylant over the resin free amino groups.

At the end of the synthesis, the dry peptide-resins were individuallytreated with 20 mL of the cleavage mixture, 88% TFA, 5% phenol, 2%triisopropylsilane and 5% water (Sole, N. A. and G. Barany, 1992, J.Org. Chem. 57:5399-5403) for 1.5 hours at room temperature. Each resinwas filtered and the solution was added to cold methyl-t-butyl ether inorder to precipitate the peptide. After centrifugation, the peptidepellets were washed with fresh cold methyl-t-butyl ether to remove theorganic scavengers. The process was repeated twice. Final pellets weredried, resuspended in H₂O, 20% acetonitrile, and lyophilized.

The synthesis of peptide OXM54 was performed by dissolving the thiolcontaining OXM peptide precursor (SEQ ID NO: 41) in TrisHCl 0.1M pH 8,guanidinium chloride 6M. A 10 molar excess of iodoacetamide was added.After 1 hour incubation, the peptide solution was purified by HPLC.

The synthesis of peptide OXM55 was performed by dissolving the thiolcontaining OXM peptide precursor (SEQ ID NO: 64) in TrisHCl 0.1M pH 8,guanidinium chloride 6M. A 10 molar excess of iodoacetamide was added tothis solution. After 1 hour incubation, the peptide solution waspurified by HPLC.

The crude peptides were purified by reverse-phase HPLC usingsemi-preparative Waters RCM Delta-Pak™ C⁻⁴ cartridges (40×200 mm, 15 um)and using as eluents (A) 0.1% TFA in water and (B) 0.1% TFA inacetonitrile. The following gradient of eluent B was used: 20%-20% over5 min and 20%-35% over 20 min, flow rate 80 mL/min. Analytical HPLC wasperformed on a Phenomenex, Jupiter C₄ column (150×4.6 mm, 5 μm) with thefollowing gradient of eluent B: 20%-40% B (in 20 min)-80% (in 3 min),flow rate 1 mL/min. The purified peptide was characterized byelectrospray mass spectrometry on a Micromass LCZ platform.

Example 2 PEGylation of Oxyntomodulin (OXM) Analogs

PEGylation reactions were run under conditions permitting thioester bondformation. The PEGylated OXM peptide was then isolated usingreverse-phase HPLC or ion exchange chromatography and size exclusionchromatography (SEC). PEGylated OXM analogs were characterized usingRP-HPLC, HPLC-SEC and MALDI-T of Mass Spectrometry.

OXM33, 34, 35, 36 and 54 peptides were synthesized from thethiol-containing OXM peptide precursor (SEQ ID NO: 41) to producederivatives with PEG covalently attached via a thioether bond.

Synthesis of OXM433

10 mg of peptide precursor (2.2 μmoles) were dissolved in 0.2 mL ofHEPES 0.1M pH 7.3, Guanidinium Chloride 6M, 2 mM EDTA. 22 mg ofMPEG-MAL-5000 (NEKTAR 2F2MOH01) (4.4 μmoles) dissolved in 0.4 mL HEPES0.1M, pH 7.3 (1:2 mole/mole ratio of peptide to PEG) was added to thissolution. After 1 hour incubation, the PEGylated peptide was purified byRP-HPLC and characterized by MALDI-T of.

Synthesis of OXM34

10 mg of peptide precursor (2.2 μmoles) were dissolved in 0.2 mL ofHEPES 0.1M pH 7.3, Guanidinium Chloride 6M, 2 mM EDTA. 80 mg ofMPEG-MAL-20K (NEKTAR 2F2M0P01) (4.0 μmoles) dissolved in 0.5 mL HEPES0.1M, pH 7.3 (1:1.8 mole/mole ratio of peptide to PEG) was added to thissolution. After 1 hour incubation, the PEGylated peptide was purified byRP-HPLC and characterized by MALDI-T of.

Synthesis of OXM35

10 mg of peptide precursor (0.92 μmoles) were dissolved in 0.4 mL ofHEPES 0.1M pH 7.3, Guanidinium Chloride 6M, 2 mM EDTA. 70 mg ofMPEG2-MAL-40K (NEKTAR 2D3Y0T01) (1.7 μmoles) dissolved in 0.8 mL HEPES0.1M, pH 7.3 in a 1:1.8 mole/mole ratio of peptide to PEG was added tothis solution. After 1 hour incubation, the PEGylated peptide waspurified by RP-HPLC and characterized by MALDI-T of.

The control peptide OXM54, was prepared by incubating the thiolcontaining peptide precursor with 10 eq. of iodoacetamide in 0.1 MTrisHCl pH 7.5, 6M guanidinium chloride. After 30 minutes incubation thepeptide was purified by RP-HPLC and characterized by electrospray massspectrometry.

The peptides OXM 56, 57, 58 were synthesized from the thiol containingOXM peptide precursor (SEQ ID NO: 64) to produce derivatives with PEGcovalently attached via a thioether bond.

Synthesis of OXM56

5 mg of peptide precursor (1.1 μmoles) were dissolved in 0.2 mL of HEPES0.1M pH 7.3. 57 mg of MPEG-MAL-5000 (NEKTAR 2F2MOH01) (11.4 μmoles)dissolved in 0.4 mL HEPES 0.1M, pH 7.3 (1:10 mole/mole ratio of peptideto PEG) was added to this solution. After 1 hour incubation, thePEGylated peptide solution was acidified to 1% acetic acid and purifiedby cation exchange chromatography (DCC) on fractogel TSK CM-650S with alinear gradient of NaCl in sodium acetate 50 mM pH 4.8. The DCC purifiedPEGylated-peptide was further purified by size-exclusion chromatography(SEC) and characterized by MALDI-T of.

Synthesis of OXM57

10 mg of peptide precursor (2.2 μmoles) were dissolved in 0.2 mL ofDMSO. 50 mg of MPEG-MAL-20K (NEKTAR 2F2M0P01) (2.5 μmoles) dissolved in0.6 mL HEPES 0.1M pH 7.3, 0.3M TRIS(2-carboxy-ethyl)phosphine with a1:1.13 mole/mole ratio of peptide to PEG was added to this solution.After 1 hour incubation, the PEGylated peptide was purified by RP-HPLCand characterized by MALDI-T of.

Synthesis of OXM58

10 mg of peptide precursor (0.92 μmoles) were dissolved in 0.4 mL ofHEPES 0.1M pH 7.3, Guanidinium Chloride 6M, 2 mM EDTA. 70 mg ofMPEG2-MAL-40K (NEKTAR 2D3Y0T01) (1.7 μmoles) dissolved in 0.8 mL HEPES0.1M, pH 7.3 (1:1.8 mole/mole ratio of peptide to PEG) was added to thissolution. After 1 hour incubation, the PEGylated peptide was purified byRP-HPLC and characterized by MALDI-T of.

The control peptides OXM102, OXM112, and OXM116 were prepared byincubating the thiol-containing peptide precursor with 10 eq. ofiodoacetamide in 0.1 M TrisHCl pH 7.5, 6M guanidinium chloride. After 30minutes incubation the peptide was purified by RP-HPLC and characterizedby electrospray mass spectrometry.

Synthesis of OXM103, OXM105, OXM107, OXM113

10 mg of the corresponding peptide precursors (2.26 μmoles) weredissolved in 2 mL of urea 8M, HEPES 0.1M pH 7.3, 2 mM EDTA. 109 mg ofMPEG2-MAL-40K (NEKTAR 2D3Y0T01) (2.71 μmoles) dissolved in H₂O (1:1.2mole/mole ratio of peptide to PEG) was added to this solution. After 1hour incubation, the PEGylated peptide solution was acidified to 1%acetic acid and purified by cation exchange chromatography (IXC) on TSKCM-650S with a linear gradient of NaCl in sodium acetate 50 mM pH 4.8.The DCC purified PEGylated-peptide was further purified by SEC andcharacterized by MALDI-T of.

Synthesis of OXM109

10 mg of the corresponding peptide precursors (2.25 μmoles) weredissolved in 2 ml urea 8M, HEPES 0.1M pH 7.3, 2 mM EDTA. 108 mg ofMPEG2-MAL-40K (NEKTAR 2D3Y0T01) (2.7 μmoles) dissolved in 2 mL H₂O(1:1.2 mole/mole ratio of peptide to PEG) was added to this solution.After 1 hour incubation, the PEGylated peptide solution was acidified to1% acetic acid and purified by cation exchange chromatography (DCC) onTSK CM-650S with a linear gradient of NaCl in sodium acetate 50 mM pH4.8. The IXC purified PEGylated-peptide was further purified by SEC andcharacterized by MALDI-T of.

Synthesis of OXM117

10 mg of the corresponding peptide precursors (2.19 μmoles) weredissolved in 2 mL of urea 8M, HEPES 0.2M pH 6.5, 2 mM EDTA. 105 mg ofMPEG₂-MAL-40K (NEKTAR 2D3Y0T01) (2.63 μmoles) dissolved in H₂O (1:1.2mole/mole ratio of peptide to PEG) was added to this solution. After 1hour incubation, the PEGylated peptide solution was acidified to 1%acetic acid and purified by cation exchange chromatography (DCC) on TSKCM-650S with a linear gradient of NaCl in sodium acetate 50 mM pH 4.8.The DCC purified PEGylated-peptide was further purified by SEC andcharacterized by MALDI-T of.

Synthesis of OXM125

10 mg of the corresponding peptide precursors (2.26 μmoles) weredissolved in 2 mL of Urea 8M, HEPES 0.25M pH 6.5, 2 mM EDTA. 105 mg ofMPEG₂-MAL-40K (NEKTAR 2D3Y0T01) (2.71 μmoles) dissolved in H₂O (1:1.2mole/mole ratio of peptide to PEG) was added to this solution. After 1hour incubation, the PEGylated peptide solution was acidified to 0.2%formic acid pH 2.8 and purified by cation exchange chromatography (IXC)on TSK SP-5PW with a linear gradient of NaCl in formic acid 0.2%. TheDCC purified PEGylated-peptide was further purified by SEC andcharacterized by MALDI-T of.

Synthesis of OXM129

10 mg of the corresponding peptide precursors (2.26 μmoles) weredissolved in 2 mL of Urea 8M, HEPES 0.25M pH 6.5, 2 mM EDTA. 109 mg ofMPEG₂-MAL-40K (NEKTAR 2D3Y0T01) (2.72 μmoles) dissolved in H₂O (1:1.2mole/mole ratio of peptide to PEG) was added to this solution. After 1hour incubation, the PEGylated peptide solution was acidified to 0.2%formic acid and purified by cation exchange chromatography (DCC) on TSKSP-5PW with a linear gradient of NaCl in formic acid 0.2%. The DCCpurified PEGylated-peptide was further purified by SEC and characterizedby MALDI-T of.

Example 3 Design and Synthesis of Peptide Sequences

The biological activity of different PEG sizes (mPEG)5 kDa, (mPEG)20kDa, (mPEG)₂40 kDa and (mPEG)₂60 kDa was compared in a series ofexperiments which demonstrated that the optimal PEG size to confer themaximal and durable activity in the mice is generally 40 kDa.

The peptide OXM103 (Aib (α) at position 2) was designed to exploreposition 20 within the oxyntomodulin sequence as the site forconjugation with (mPEG)₂40 kDa. OXM102 is a control peptide (CH₂CONH₂),in which the side-chain thiol (of cysteine at position 20) was reactedwith iodoacetamide.

The peptide OXM105 (Aib (α) at position 2) was designed to exploreposition 21 within the oxyntomodulin sequence, as the site forconjugation with (mPEG)₂40 kDa. OXM104 is a control peptide (CH₂CONH₂),in which the side-chain thiol (of cysteine at position 21) was reactedwith iodoacetamide.

The peptide OXM107 (Aib (α) at position 2, Met(O) at position 27) wasdesigned to explore position 24 within the oxyntomodulin sequence, asthe site for conjugation with (mPEG)₂40 kDa. OXM106 is a control peptide(CH₂CONH₂), in which the side-chain thiol (of cysteine at position 24)was reacted with iodoacetamide.

The peptide OXM109 (Aib (α) at position 2,) was designed to exploreposition 28 within the oxyntomodulin sequence, as the site forconjugation with (mPEG)₂40 kDa. OXM108 is a control peptide (CH₂CONH₂),in which the side-chain thiol (of cysteine at position 28) was reactedwith iodoacetamide.

The peptide designated OXM141 has a Gln to Asp mutation at position 3,which confers specific selectivity to the GLP1-receptor. The peptideOXM141, which has a Gln to Asp mutation at position 3, Met(O) atposition 27, and two conjugation sites at position 20 and 38, wasdesigned to explore the potential of having a peptide conjugated withboth a cholesterol group at C38 and PEG at position C20.

The peptide OXM142 (Aib (α), Met(O) at position 27, and two conjugationsites at position 20 and 38) was designed to explore the potential ofhaving a peptide conjugated with both a, cholesterol group at C38 andPEG at position C20.

FIG. 14 summarizes the in vitro activity data for GLP1R and GCGreceptors (also showing the GLP-1R and GLGR specificities) in tabularform. The (mPEG)₂40 kDa conjugate at position C20 retains activity onboth receptors, therefore OXM103 as is referred to as a +/+ analog,while all the other (mPEG)₂40 kDa conjugates lose potency at the GCGreceptor. In particular there is a between 2-3 orders of magnitudeselectivity towards the GLP-1 receptor over the Gcg receptor for the(mPEG)₂40 kDa conjugates at position 38, 24 and 28. Therefore, theanalogs OXM107 and 109 are referred to as +/0 analogs.

FIG. 15 shows the in vivo activity of the (mPEG)₂40 kDa conjugates onfood intake and body weight loss on DIO mice. Ad libitum fed, DIO (˜51 geach), male C57BL/6 mice were dosed i.p. with either vehicle (water) orOxm analogs 99, 103, 105, 107, and 109 ˜30 min prior to the onset of thedark phase. Food intake measured ˜2 h and 18 h (overnight) later on day1 and 26 and 42 h (overnight) later on day 2. *P<0.05 vs. vehicle, n=5-6per group).

The peptide OXM110 (D-Ser (s) at position 2,) was designed to exploreposition 38 within the oxyntomodulin sequence, as the site forconjugation with a lipid such as a palmitoyl group. The palmitoyl groupwas acylated to the ε-amino group of a lysine added at the C-terminus ofthe oxyntomodulin sequence.

The peptide OXM113 (desamino-His (ΔNH₂—H) at position 1, Aib (α) atposition 2, Met(O) at position 27, conjugation site 38) was designed toexplore the potential of substituting the wild type (wt) His with adesamino-His at position 1 for protection from DPPIV proteolysis. OXM113is the (mPEG)₂40 kDa conjugate, OXM114 is the cholesteryl conjugate andOXM112 is a control peptide (CH₂CONH₂), in which the side-chain thiol(of cysteine at position 38) was reacted with iodoacetamide). (OXM111 isthe thiolated peptide precursor).

The peptides OXM117 and OXM118 (Aib (α) at position 2, Gln to Aspmutation at position 3, Met(O) at position 27, conjugation site 38) weredesigned to explore the potential of substituting the wild type Gln withan Asp at position 3. As mentioned above, this mutation confers specificselectivity towards the GLP-1R. OXM117 is the (mPEG)₂40 kDa conjugateand the OXM118 is the cholesteryl conjugate. (OXM116 is the thiolatedpeptide precursor).

The peptide OXM121 (Aib (α) at position 2, Met(O) at position 27,conjugation site 11) was designed to explore position 11 within theoxyntomodulin sequence as the site for the conjugation. OXM121 is the(mPEG)₂40 kDa conjugate, OXM119 is the thiolated peptide precursor andOXM120 is a control peptide (CH₂CONH₂), in which the side-chain thiol(of cysteine at position 11) was reacted with iodoacetamide.

The peptide OXM124 (Aib (α) at position 2, Met(O) at position 27,conjugation site 12) was designed to explore position 12 within theoxyntomodulin sequence as the site for conjugation. OXM124 is the(mPEG)₂40 kDa conjugate, OXM122 is the thiolated peptide precursor andOXM123 is a control peptide (CH₂CONH₂), in which the side-chain thiol(of cysteine at position 12) was reacted with iodoacetamide.

The peptide OXM125 (Aib (α) at position 2, Gln to Asp mutation atposition 3, Met(O) at position 27, conjugation site 20) was designed toexplore position 20 within the oxyntomodulin sequence as the site forconjugation of (mPEG)₂40 kDa as well as the Gln to Asp substitution atposition 3 that confers selectivity towards the GLP-1R

The peptide OXM127 (Aib (α) at position 2, Met(O) at position 27,conjugation site 22) was designed to explore position 22 within theoxyntomodulin sequence as the site for conjugation for cholesterol.OXM126 is the thiolated peptide precursor).

The peptide OXM129 (desamino-His (ΔNH₂—H) at position 1, Aib (α) atposition 2, Gln to Asp mutation at position 3, Met(O) at position 27,conjugation site 20) was designed to explore the potential of thefollowing combinations: substituting the wt His with desamino-His atposition 1 for protection from DPPIV proteolysis, the Gln to Aspsubstitution at position 3 that confers selectivity towards the GLP-1R,and conjugation at position 20.

The peptide OXM134 (Aib (α) at position 2, Met(O) at position 27,conjugation site 20) is a(mPEG)₂40 kDa conjugate similar to the potent+/+ OXM103 analogue that only differs for the methionine substitution tomethionine sulfoxide. This peptide is conjugated at the position 20therefore displaying a +/+ pattern for GLP1R/GcgR selectivity.

Example 4 C-Terminal Truncated Analogs or GcgK Analogs

C-Terminal Truncated Analogs

A series of peptide OXM C-terminal truncated analogs were designed. Ananalysis of in vitro activity on both the GLP1 receptor (GLP1R) and theGcg receptor (GcgR) demonstrated that the OXM sequence can be truncatedat the C-terminus to have only one extra lysine residue with respect towt glucagon, yielding a peptide such as OXM93 that is extremely potenton both the GLP1R and GcgR. The potency of OXM93 is at least one order(and possibly two) of magnitude higher than that of wt OXM. Becausethere is only one extra Lys residue with respect to Gcg, this new classis referred to as GcgK analogs. FIG. 16 illustrates in vitro potencydata for the C-terminal truncated analogs acting at the GLP1 and GCGreceptors in tabular form.

C-terminal truncated analogs which are selective GcgK Analogs

Peptides OXM130 and OXM131 were designed to confirm the in vitroanalysis and introduce other mutations that are known to conferstability and suitable properties. OXM130 and OXM131 are C-terminaltruncated analogues of the same length as OXM93. OXM130 has Aib (α) atposition 2, while OXM131 has Aib (α) at position 2 and a Gln to Aspmutation at position 3 to confer selectivity for the GLP1R.

The following PEGylated analogs were designed based on these truncatedsequences:

The peptide OXM136 (Aib (α) at position 2, Met(O) at position 27,conjugation site 20) is the (mPEG)₂40 kDa conjugate. From prior studies(see above) it is known that the choice of PEG conjugation at position20 allows for activity on both receptors GLP1R and GcgR, thereforeOXM136 would be defined as the prototype Gcg +/+ analogue. (OXM135 isthe thiolated peptide precursor).

The peptide OXM138 (Aib (α) at position 2, Gln to Asp mutation atposition 3, Met(O) at position 27, conjugation site 20) was designed toexplore the potential of the following combinations: the Gln to Aspsubstitution at position 3 that confers selectivity towards the GLP-1R;conjugation at position 20 and mutating the wt Gln to Asp at position 3.Therefore OXM137 would be defined as the prototype GcgK +/0 analog(OXM137 is the thiolated peptide precursor).

FIG. 17 provides in vitro potency data at the GLP1 and GCG receptors forselect PEGylated OXM analogs.

Example 5 Peptides with Alternative PEG Moieties

The peptide OXM145 (Aib (α) at position 2, Gln to Asp mutation atposition 3, Met(O) at position 27, conjugation site 38 with Y Shape PEGMaleimide from JenKem Therapeutics) was produced to explore thepotential of using alternative branched 40 kDa PEG moieties. This is theconjugate obtained using a (mPEG)₂40 kDa from JenKem Therapeutics(Y-Shaped PEG Maleimide, MW40K, item number Y-MAL-40K and its structureis shown below:

The peptide OXM146 is characterized by Aib (α) at position 2, Gln to Aspmutation at position 3, Met(O) at position 27, conjugation site 38 withSUNBRIGHT GL2-400MA Maleimide from NOF corporation. This is theconjugate obtained using a (mPEG)₂40 kDa from NOF Corporation (SUNBRIGHTGL2-400MA Maleimide) and its structure is shown below:

The peptide OXM151 (Aib (α) at position 2, Gln to Asp mutation atposition 3, Arg to Glu mutation at position 17, Arg to Ala mutation atposition 18, Met(O) at position 27, conjugation site 38) was designed toconfer in vivo increased stability to the peptide sequence.

A detailed study was undertaken in which OXM139 was incubated in PBScontaining 10% of mouse or human plasma at 37° C. Sample preparation wasaccomplished by mixing 1 μL of test solution and 1 μL of matrix(α-cyano) directly on the sample plate. After crystallization T ofspectrum was collected: Time points at 30, 60, 120 and 720 minutes wereanalyzed and compared with the same time points of the control testsolutions. Within the peptide sequence, the bond between Arg17 and Arg18has been identified to be a primary hydrolysis site. The bond betweenArg18 and Ala19 was also identified as a secondary site of hydrolysis,so it was decided to introduce mutations at sites 17 and 18.Specifically, Arg 17 was mutated to Glu and Arg 18 was mutated to Ala.The peptide OXM153 is the N-ethyl maleimide analog of OXM151. Thepeptide OXM154 is the conjugate obtained using a (mPEG)₂40 kDa from NOFCorporation (SUNBRIGHT GL2-400MA Maleimide).

The peptide OXM152 spans residues 1 to 16 of the Oxyntomodulin sequence(Aib (α) at position 2, Gln to Asp mutation at position 3, Ttds atposition 17 as a spacer, Cys at position 18 for conjugation) and is apeptide that can be conjugated to a carrier protein to raise antibodiesspecific against the 1-16 sequence.

The peptide OXM155 (Aib (α) at position 2, Gln to Asp mutation atposition 3, Met(O) at position 27, Ttds at position 38, 5 glutamicresidues a position 39-43, Cys at position 44 for conjugation) isanother peptide that can be conjugated to a carrier protein to raiseantibodies. The addition of glutamic acids at the C terminus is neededfor pI modulation that will enable conjugation to the carrier protein,as described in “A Method to Make a Peptide-Carrier Conjugate with aHigh Immunogenicity,” Provisional Application Ser. No. 60/530,867, filedon Dec. 18, 2003, and herein incorporated by reference in its entirety.

Example 6 New Truncated Analogs or GcgK Analogs

The peptide OXM143 (Aib (α), Met(O) at position 27, conjugation site 20)is the N-ethyl maleimide analog of OXM135.

The peptide OXM144 (Aib (α), Gln to Asp mutation at position 3, Met(O)at position 27, and conjugations site at position 20 is the N-ethylmaleimide analog of OXM137.

The peptide OXM147 (Aib (α) at position 2, conjugation site 20) wasdesigned to have a native Methionine residue at position 27 within theGcgK analog series having the conjugation site at C20. The rationale forthis design was that a peptide analog with a native methionine is moreactive on the glucagon receptor.

The peptide OXM148 (Aib (α) at position 2, conjugation site 20) is theN-ethyl maleimide analog of OXM147.

The peptide OXM149 (Aib (α), Gln to Asp mutation at position 3, Met(O)at position 27, conjugations site at position 20 and D-Lysinereplacement at position 30) was designed to provide protection in vivofrom enzymatic degradation of the C-terminus of the peptide. The peptideOXM150 is the N-ethyl maleimide analog of OXM149.

A similar study of stability was performed on a GcgK series analogOXM144 to determine the primary sites of hydrolysis by incubation withPBS, containing 10% of either mouse or human plasma. In this instance,the bond between Arg17 and Arg18 was identified as a primary hydrolysissite. Also the bond between Arg18 and Ala19 was identified as asecondary site of hydrolysis. For this reason, it was decided tointroduce mutations at the sites 17 and 18 also in the GcgK analogseries.

Synthesis of OXM145

54 mg of the corresponding peptide precursors (11.8 μmoles) weredissolved in 2 mL of Urea 8M, HEPES 0.2M pH 6.5, 2 mM EDTA. 569 mg (14.2μmoles) of Y-Shaped PEG Maleimide, MW40K (JenKem Technology, item numberY-MAL-40K) dissolved in H₂O (1:1.2 mole/mole ratio of peptide to PEG)was added to this solution. After 1 hour incubation, the PEGylatedpeptide solution was acidified to 0.2% formic acid pH 2.8 and purifiedby cation exchange chromatography (IXC) on TSK SP-5PW with a lineargradient of NaCl in formic acid 0.2%. The IXC purified PEGylated-peptidewas further purified by SEC and characterized by MALDI-T of.

Synthesis of OXM146

55 mg of the corresponding peptide precursors (12 moles) were dissolvedin 2 mL of Urea 8M, HEPES 0.2M pH 6.5, 2 mM EDTA. 531 mg (13.2 μmoles)of SUNBRIGHT GL2-400MA Maleimide, (NOF Corporation) dissolved in H₂O(1:1.1 mole/mole ratio of peptide to PEG) was added to this solution.After 1 hour incubation, the PEGylated peptide solution was acidified to0.2% formic acid pH 2.8 and purified by cation exchange chromatography(IXC) on TSK SP-5PW with a linear gradient of NaCl in formic acid 0.2%.The DCC purified PEGylated-peptide was further purified by SEC andcharacterized by MALDI-T of.

Example 7 Measurement of GLP-1 Receptor (GLP-1R) Signaling Using aCyclic AMP (cAMP) Homogenous Time Resolved Fluorescence (HTRF) Assay andEvaluation of Resistance to DP-IV

Chinese hamster ovary (CHO) cell lines stably transfected with a mutantform of the human GLP-1R with bioactivity similar to that of the nativereceptor were maintained in complete Iscove's Modified Dulbecco's Medium(IMDM) media containing fetal bovine serum (FBS),penicillin-streptomycin, hypoxanthine-thymidine and G418. A homogenoustime resolved fluorescence (HTRF) assay for GLP-1 receptor activationwas used to measure cAMP accumulation in transfected cells uponincubation with peptides of this invention following the manufacturer'sinstructions (Cis Bio), with the exception that cells were incubatedwith ligand, XL-665 and anti-cAMP cryptate at 37° C. The assay wasconducted in a 96 half-well plate format and the plate was read using aPerkin Elmer Envision plate reader. For polypeptides and polypeptidefragments/derivatives of this invention, “activation” of the GLP-1receptor in a cAMP HTRF assay is induction of a maximal activity that isat least about 60% and up to about 200% of the maximal activity inducedby the native human OXM sequence with a relative potency of at least0.04% up to about 1000%. “Relative potency” is the EC₅₀ of native humanOXM divided by the EC₅₀ of the polypeptide of the invention, multipliedby 100. “EC₅₀” is the concentration of a polypeptide at which 50% of themaximal activity is achieved.

To measure resistance to DP-IV cleavage, a 5 μM solution of peptide waspre-incubated with 10 nM of recombinant soluble human DP-IV in 100 μlassay buffer (10 mM HEPES, pH 7.5, 0.05% BSA) at 37° C. for 2 hours.Activation of hGLP-1R was subsequently measured using the Cis Bio HTRFcAMP assay and compared to control peptides pre-incubated at 37° C. for2 hours in the absence of DP-W. For polypeptides and theirfragments/derivatives of this invention, “resistance to DP-IV” in thisexperiment is defined as a potency ratio of 0.1 up to 35, where “potencyratio” is the EC₅₀ of a peptide preincubated with DP-IV divided by theEC₅₀ of the same polypeptide of the invention preincubated withoutDP-IV. (FIG. 2)

Example 8 Measurement of Glucagon Receptor (GcgR) Signaling Using aCyclic AMP Flashplate Assay

CHO cells expressing the cloned human glucagon receptor (CHO-hGCGR)(Cascieri et al., J. Biol. Chem. (1999), 274, 8694-8697) were maintainedin IMDM supplemented with 10% FBS, 1 mM L-glutamine,Penicillin-Streptomycin (100 U/ml), and G418 (500 μg/ml) cAMP levels intransfected cells upon incubation with peptides of this invention weredetermined with the aid of a Flashplate assay (SMP-004B, Perkin ElmerLife Sciences) following the manufacturer's instructions. The cellstimulation was stopped by addition of an equal amount of a detectionbuffer containing cell lysis agent and ¹²⁵I-labeled cAMP tracer. The¹²⁵I-cAMP bound to the plate was determined using a liquid scintillationcounter and used to quantitate the amount of cAMP present in eachsample. For polypeptides and polypeptide fragments/derivatives of thisinvention, “activation” of the Gcg receptor in a cAMP Flashplate assayis induction of a maximal activity that is at least about 60% up toabout 200% of the maximal activity induced by the native glucagonpeptide with a relative potency of at least 0.04% up to about 10000%.“Relative potency” is the EC₅₀ of native glucagon (EC₅₀=70 pM) dividedby the EC₅₀ of the polypeptide of the invention, multiplied by 100.“EC₅₀” is the concentration of a polypeptide at which 50% of the maximalactivity is achieved. Native porcine OXM activated the glucagon receptorwith an EC₅₀ of 2.4 nM in this assay.

Example 9 Effect on Blood Glucose Excursion During an IntraperitonealGlucose Tolerance Test (IPGTT) in Lean Mice

Male C57BL/6N mice were distributed by weight into treatment groups andfasted approximately 5 hours prior to the start of the study. Baseline(t=−30 min) blood glucose concentration was determined by glucometerfrom tail nick blood. Animals were then injected intraperitoneally(i.p.) with vehicle (saline) or a polypeptide of the invention (0.01-10mg/kg). Blood glucose concentration was measured 30 minutes aftertreatment (t=0 min) and mice were then challenged i.p. with dextrose (2g/kg, 10 mL/kg). One group of vehicle-treated mice was challenged withnormal saline as a negative control. Blood glucose levels weredetermined from tail bleeds taken 20, 40, 60 and 120 min after dextrosechallenge. The blood glucose excursion profile from t=0 to t=120 min wasused to integrate an area under the curve (AUC) for each treatment.Percent inhibition of glucose excursion for each treatment group wascalculated from the AUC data normalized to the water-challenged controlsas per the formula:

${{\%\mspace{14mu}{inhibition}} = {\frac{{AUC}_{dex} - {AUC}_{peptide}}{{AUC}_{dex} - {AUC}_{saline}} \times 100}},$whereAUC_(dex)=average AUC for vehicle-treated dextrose-challenged animals,AUC_(peptide)=average AUC for peptide-treated dextrose-challengedanimals, andAUC_(saline)=average AUC for vehicle-treated saline-challenged animals.

Incretin activity of a polypeptide of the invention in IPGTT ismanifested as a dose-dependent increase in percent inhibition of glucoseexcursion, reaching at least 30% at the 10 mg/kg dosage. (FIG. 2)

Example 10 Acute Effects on Food Intake and Body Weight in Lean Mice

Approximately 3-month-old, ad libitum fed, male C57BL/6N mice wereweighed and either vehicle (water) or OXM2 or OXM3 dissolved in vehiclewas administered by i.p. injection ˜30 min. prior to the onset of thedark phase of the light cycle. A pre-weighed aliquot of rodent chow(Teklad 7012) was provided in the food hopper of the wire cage top ˜5min. prior to the onset of the dark phase of the light cycle and weighed2 and 18 hr (overnight) after the onset of the dark phase of the lightcycle. Absolute changes in food intake were calculated for each animalby subtracting the amount of food remaining in the food hopper at thespecified time points from that of the corresponding originalpre-weighed aliquot. Absolute changes in body weight were calculated foreach animal by subtracting the body weight of the animal prior to dosingfrom that of the corresponding animal at the specified time points. Allvalues were reported as mean±SEM and peptide treatment groups wereanalyzed by the two-tailed unpaired Student's t test with reference tovehicle-treated animals. Reductions in food intake at any time pointand/or in overnight body weight gain are considered to be statisticallysignificant for P values ≦0.05 and denotes efficacy of the correspondingOXM polypeptide (OXM2 or OXM3) in this model. (FIG. 3)

Example 11 Enhancement of Glucose-Stimulated Insulin Secretion in Mice

The in vitro potencies of native OXM in mediating insulin secretion at16 mmol/l glucose were evaluated by measuring glucose-stimulated insulinsecretion (GSIS) at 16 mmol/l glucose in the presence of increasingconcentrations of the native OXM peptide in islets from wild typeC57BL/6 mice and in MIN6c4 cells, a mouse insulinoma cell line withrobust GSIS activity (Minami K, et al 2000 Am J Physiol EndocrinolMetab. 279:E773-E781). Pancreatic islets of Langerhans were isolatedfrom the pancreas of normal C57BL/6J mice (Jackson Laboratory, Maine) bycollagenase digestion and discontinuous Ficoll gradient separation, amodification of the original method of Lacy and Kostianovsky (Lacy etal., Diabetes 16:35-39, 1967). The islets were cultured overnight inRPMI 1640 medium (11 mM glucose) before GSIS assay. To measure GSIS,islets were first preincubated for 30 minutes in the Krebs-Ringerbicarbonate (KRB) buffer with 2 mM glucose (in Petri dishes). The KRBmedium contains 143.5 mM Na⁺, 5.8 mM K⁺, 2.5 mM Ca²⁺, 1.2 mM Mg²⁺, 124.1mM 1.2 mM PO₄ ^(3″, 1.2) mM SO₄ ²⁺, 25 mM CO₃ ²⁻, 2 mg/ml bovine serumalbumin (pH 7.4). The islets were then transferred to a 96-well plate(one islet/well) and incubated at 37° C. for 60 minutes in 200 μl of KRBbuffer with 2 or 16 mM glucose, along with other agents to be testedsuch as GLP-1 and OXM (Zhou et al., J. Biol. Chem. 278:51316-51323,2003). Insulin was measured in aliquots of the incubation buffer byELISA with a commercial kit (ALPCO Diagnostics, Windham, N.H.). Theinsulin secretion in the MIN6c4 cell was measured in cell seeded in96-well plate in similar manner.

As shown in FIG. 4, the native OXM significantly enhanced GSIS in bothmouse islets and the MIN6c4 cell. The EC50 of OXM on GSIS was about 2.9nM in murine islets (FIG. 4A) and 155 pM respectively, FIG. 4B). NativeGLP-1 was used as the positive control in this experiment. The maximalGSIS effects of the two peptides were similar in both islets and in MIN6cells.

OXM activates both GLP-1R and GCG-R heterologously expressed in CHOcells as described in Example 7 and 8, and both receptors are known tobe functional in pancreatic β-cells. To discern the potential roles ofthese two G-protein coupled receptors in the incretin action of OXM, theeffects of OXM, GLP-1 and GCG on GSIS in islets from GLP-1R−/− mice(Scrocchi L A, et al. 1996 Nat Med 2:1254-1258) and age-matched WTC57BL/6 mice were examined. Consistent with previous results, all threepeptides (10 nM each) were equally efficacious in augmenting GSIS at 16mmol/l glucose from WT murine islets (FIG. 5A). GSIS and thepotentiation of GSIS by GCG were not impaired in GLP-1R−/− islets,whereas both GLP-1 and OXM were completely unable to enhance GSIS in thelatter. The involvement of GLP-1R in the incretin action of OXM was alsoindicated by antagonism of this activity by exendin-9, a widely-usedpeptide antagonist of GLP-1R. The potentiation of GSIS by OXM and GLP-1was completely blocked in WT islets by 0.5 μM exendin-9 (FIG. 5B).

The potential participation of GCG-R in the incretin action of OXM wastested by comparing peptide-mediated GSIS at 16 mmol/l glucose in isletsfrom WT and GCG-R−/− mice (Gelling, X Q, et al 2003; Proc. Natl. Acad.Sci. U.S.A. 100: 1438-1443). As described earlier, all three peptides(GLP-1, OXM and GCG, at 10 nM each) enhanced GSIS in WT islets withequal efficacy (FIG. 6A). Compared to size-matched WT islets however,insulin secretion at both 2 and 16 mM glucose was reduced by ˜2-fold inGCG-R−/− islets (FIG. 6A). Islet insulin content was also reduced inGCG-R −/− islets by >3-fold relative to WT (FIG. 6B). GCG (10 nM) didnot enhance GSIS at 16 mM glucose in GCG-R−/− islets whereas both GLP-1and OXM (10 nM) significantly increased GSIS in this assay. When datawere expressed as fractional GSIS (% insulin released relative to totalislet insulin content), the fold-increase in GSIS mediated by OXM wasreduced by only 32% (1.7 vs 2.5 fold) in GCG-R−/− islets relative to WT(FIG. 6C), whereas the fold-stimulation of GSIS by GLP-1 remained thesame (2.5 fold). In contrast, GCG did not increase fractional GSIS overbaseline (DMSO) in GCG-R−/− islets. These data suggest that GCG-R mayplay limited role in the action of OXM on GSIS.

To determine whether the glucose-lowering effect of OXM (as describedsupra and as shown in FIG. 2) was secondary to increased in vivo GSISthe effects of OXM on plasma glucose and insulin levels during an IPGTTin WT and GLP-1R−/− mice was analyzed. Mice fasted overnight werepre-dosed with 0.3 mpk (mg peptide per kg of body weight) of native OXM(i.p.) prior to glucose challenge. The GLP-1 mimetic exendin-4 (dosed at0.02 mpk i.p.) (Thorens, B, et al. 1993; Diabetes. 42: 1678-1682) wasused as a comparator in this study. As shown in FIG. 7A, both exendin-4and OXM significantly reduced glucose levels during an IPGTT in WT mice,with exendin-4 being more potent in suppressing glucose excursion. Thearea under the curve (AUC) for glucose excursion in the 0.3 mpk OXMtreated group was reduced by approximately 30% relative to the vehiclegroup [13025 f 524 versus 19928±811 mg/dl/60 min], p<0.001, n=10(vehicle) or 5 (OXM)], whereas reduction of glucose AUC in the exendin-4treated group was >60% (AUC=6601±179 mg/dl/60 min). In contrast, thesame doses of OXM and exendin-4 did not affect glucose excursion in anIPGTT in GLP-1R−/− mice (FIG. 7B).

The effects of i.p. OXM and exendin-4 on in vivo GSIS were assessed bymeasuring plasma insulin levels before (at 0 min) and after (at 10 min)glucose challenge in the IPGTT studies. OXM increased basal (0 min)plasma insulin levels 4-fold in WT mice and significantly amplified theinsulin response to i.p. glucose challenge (FIG. 7C). Similar effectswere observed with exendin-4 in WT mice. In contrast, administration ofOXM or exendin-4 to the GLP-1R−/− mice did not affect basal insulinlevels, nor did it improve the insulin response to i.p. glucosechallenge (FIG. 7D).

EXAMPLES OF PHARMACEUTICAL COMPOSITIONS

As a specific embodiment of an oral composition of a novel polypeptideof the present invention, 5 ma of a polypeptide as described by theformula

HαDGTFTSDYSKYLDSRRAQDFVQWLmNTKRNRNNIAC₁₀-CONH_(2,)is formulated with sufficient finely divided lactose to provide a totalamount of 580 to 590 mg to fill a size 0 hard gel capsule.

As another specific embodiment of an oral composition of a novelpolypeptide of the present invention, 2.5 mg of a polypeptide asdescribed by the formula

HαDGTFTSDYSKYLDSRRAQDFVQWLmNTKRNRNNIAC₁₀-CONH_(2,)is formulated with sufficient finely divided lactose to provide a totalamount of 580 to 590 mg to fill a size 0 hard gel capsule.

Other embodiments are within the following claims. While severalembodiments have been shown and described, various modifications may bemade without departing from the spirit and scope of the presentinvention.

1. A polypeptide comprising the amino acid sequence of SEQ ID NO:4,further comprising a substitution of Asp, Glu, Ile, Val, D-Ser, Met,D-Ala, Trp, Asn, Leu, or α-aminoisobutyric acid for the Ser at position2 and a pharmaceutically acceptable salt thereof.
 2. The polypeptide ofclaim 1, wherein the polypeptide further comprises one or more aminoacid substitutions selected from (a) Asp, Glu, Pro, Leu or L-norleucinefor the Gln at position 3; (b) Ala, Cys, Cys(mPEG), or Cys(cholesteryl)for the Ser at position 11; (c) Cys, Cys(mPEG), or Cys(cholesteryl) forthe Lys at position 12; (d) Ala for the Ser at position 16; (e) Cys,Cys(mPEG), or Cys(cholesteryl) for the Arg at position 17; (f) Cys,Cys(mPEG), or Cys(cholesteryl) for the Arg at position 18; (g) Cys,Cys(mPEG), or Cys(cholesteryl) for the Gln at position 20; (h) Cys,Cys(mPEG), or Cys(cholesteryl) for the Asp at position 21; (i) Cys,Cys(mPEG), or Cys(cholesteryl) for the Gln at position 24; (j) Met(O),Val, norleucine, alanine, α-aminoisobutyric acid or O-methyl-homoserinefor the Met at position 27; (k) Cys, Cys(mPEG), or Cys(cholesteryl) forthe Asn at position 28; (l) Cys, Cys(mPEG), or Cys(cholesteryl) for theThr at position 29; (m) Cys, Cys(mPEG), or Cys(cholesteryl) for the Ileat position 36; or (n) Cys, Cys(mPEG), or Cys(cholesteryl) for the Alaat position
 37. 3. The polypeptide of claim 1, wherein the polypeptidefurther comprises a deletion of amino acids 31-37.
 4. The polypeptide ofclaim 1, wherein the polypeptide further comprises a Lys(Palmitoyl),Cys, Cys(mPEG), or Cys(cholesteryl) at the carboxy terminus.
 5. Thepolypeptide of claim 1, wherein the polypeptide comprisesα-aminoisobutyric acid for the Ser at position 2, Asp for the Gln atposition 3, Met(O) for the Met at position 27, and a Cys(mPEG) at thecarboxy terminus.
 6. The polypeptide of claim 1, wherein the polypeptidecomprises α-aminoisobutyric acid for the Ser at position 2, Asp for theGln at position 3, Met(O) for the Met at position 27, and aCys(cholesteryl) at the carboxy terminus.
 7. The polypeptide of claim 1,wherein the polypeptide comprises D-Ser for the Ser at position 2 and aLys(palmitoyl) at the carboxy terminus.
 8. The polypeptide of claim 1,wherein the polypeptide is selected from a polypeptide comprising theamino acid sequence of SEQ ID NO:114, SEQ ID NO:120, or SEQ ID NO:121.9. A pharmaceutical composition comprising the polypeptide of claim 1and a pharmaceutically suitable carrier.
 10. A method for reducing bodyweight in a subject in need thereof comprising the step of administeringto the subject a polypeptide comprising the amino acid sequence of SEQID NO:4, further comprising a substitution of Asp, Glu, lie, Val, D-Ser,Met, D-Ala, Trp, Asn, Leu, or α-aminoisobutyric acid for the Ser atposition 2 and a pharmaceutically acceptable salt thereof.
 11. Themethod of claim 10, wherein the polypeptide further comprises one ormore amino acid substitutions selected from (a) Asp, Glu, Pro, Leu orL-norleucine for the Gln at position 3; (b) Ala, Cys, Cys(mPEG), orCys(cholesteryl) for the Ser at position 11; (c) Cys, Cys(mPEG), orCys(cholesteryl) for the Lys at position 12; (d) Ala for the Ser atposition 16; (e) Cys, Cys(mPEG), or Cys(cholesteryl) for the Arg atposition 17; (f) Cys, Cys(mPEG), or Cys(cholesteryl) for the Arg atposition 18; (g) Cys, Cys(mPEG), or Cys(cholesteryl) for the Gln atposition 20; (h) Cys, Cys(mPEG), or Cys(cholesteryl) for the Asp atposition 21; (i) Cys, Cys(mPEG), or Cys(cholesteryl) for the Gln atposition 24; (j) Met(O), Val, norleucine, alanine, α-aminoisobutyricacid or O-methyl-homoserine for the Met at position 27; (k) Cys,Cys(mPEG), or Cys(cholesteryl) for the Asn at position 28; (l) Cys,Cys(mPEG), or Cys(cholesteryl) for the Thr at position 29; (m) Cys,Cys(mPEG), or Cys(cholesteryl) for the Ile at position 36; or (n) Cys,Cys(mPEG), or Cys(cholesteryl) for the Ala at position
 37. 12. Themethod of claim 10, wherein the polypeptide further comprises a deletionof amino acids 31-37.
 13. The method of claim 10, wherein thepolypeptide further comprises a Lys(Palmitoyl), Cys, Cys(mPEG), orCys(cholesteryl) at the carboxy terminus.
 14. The method of claim 10,wherein the polypeptide comprises α-aminoisobutyric acid for the Ser atposition 2, Asp for the Gln at position 3, Met(O) for the Met atposition 27, and a Cys(mPEG) at the carboxy terminus.
 15. The method ofclaim 10, wherein the polypeptide comprises α-aminoisobutyric acid forthe Ser at position 2, Asp for the Gln at position 3, Met(O) for the Metat position 27, and a Cys(cholesteryl) at the carboxy terminus.
 16. Themethod of claim 10, wherein the polypeptide comprises D-Ser for the Serat position 2 and a Lys(palmitoyl) at the carboxy terminus.
 17. Themethod of claim 10, wherein the polypeptide is selected from apolypeptide comprising the amino acid sequence of SEQ ID NO:114, SEQ IDNO:120, or SEQ ID NO:121.
 18. The method of claim 10, wherein thetreatment lowers glucose levels in the subject.